Treating Cancer with Drug Combinations

ABSTRACT

Embodiments of the disclosure find application in the field of cancer therapy. Receptor protein kinases (RPTKs) transmit extracellular signals across the plasma membrane to cytosolic proteins, stimulating formation of complexes that regulate key cellular functions. Over half of the known tyrosine kinases are implicated in human cancers and are therefore highly promising drug targets.

CROSS-REFERENCES TO RELATES APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/877,300 filed on Oct. 7, 2015 (now allowed as U.S. Pat. No.10,668,068), the contents of which are fully incorporated herein byreference.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with government support under grant no. CA136658awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

Embodiments of the invention find application in the field of cancertherapy. Receptor protein kinases (RPTKs) transmit extracellular signalsacross the plasma membrane to cytosolic proteins, stimulating formationof complexes that regulate key cellular functions. Over half of theknown tyrosine kinases are implicated in human cancers and are thereforehighly promising drug targets.

BACKGROUND OF THE INVENTION

Protein tyrosine kinases (PTKs) mediate the reversible process oftyrosine phosphorylation, providing the signals that activate or blocksignal transduction pathways that govern cell survival decisions and assuch are tightly regulated. Genes that regulate extracellular growth,differentiation and developmental signals are commonly mutated incancers. Perhaps it is not surprising therefore that PTKs comprise thelargest group of dominant oncogenes. Thirty of the 58 receptor proteintyrosine kinases (RPTKs) have been implicated in human cancer(Blume-Jensen and Hunter, 2001 [1]). Less than half of the cytoplasmicprotein tyrosine kinases have been associated with tumorigenesis, duenot to a less critical role in signal transduction regulation, however,but from an experimental bias that has focused on viral counterparts togain insight into potential transforming mechanisms (Blume-Jensen andHunter, 2001 [1]).

In recent years there has been a surge in efforts to discover genescritical to cancer signaling pathways that when inhibited would providespecific anti-cancer therapies (Lu and Chu, 2008 [2]) (Sabbah et al.,2008 [3]). Trastuzumab, (Herceptin), a humanized monoclonal antibodythat specifically inhibits the HER2/neu/ErbB-2 (hereafter referred to asErbB-2) receptor tyrosine kinase, which is amplified and/orover-expressed in 25-30% of metastatic breast cancers, was the firsttargeted therapy to be approved by the FDA. As a single-agentmonotherapy, however, the primary response rate to trastuzumab is low,(12% to 34%) and the rate of primary resistance high, between 66% to 88%(Nahta and Esteva, 2006 [4]). Notably, however, the time to diseaseprogression, response rate and overall survival increase whentrastuzumab is used in combination with paclitaxel or docetaxel (Nahtaand Esteva, 2006 [4]). Indeed, recent successes in targeting moleculesintegral to survival pathways in combination with traditionalchemotherapeutics has led to significant efforts to identify new drugtargets that sensitize the breast cancer cell towards cell death(MacKeigan et al., 2005 [5]); (Call et al., 2008 [6]). Such additionaldrug targets, specific to or over-expressed in breast cancer cellscompared to normal tissues, and known to be functionally relevant, arestill needed, as are cancer-specific markers for use in detecting ordiagnosing cancer.

While a number of inhibitors have been reported, it is not clear whetherthey possess the appropriate pharmacological properties to betherapeutically useful. Therefore, there is a continued need for smallmolecule inhibitors to provide effective inhibition. Such compoundswould be extremely useful in treating the disease states whereinhibition could play a role.

SUMMARY

Embodiments of the invention find application in the field of cancertherapy. Receptor protein kinases (RPTKs) transmit extracellular signalsacross the plasma membrane to cytosolic proteins, stimulating formationof complexes that regulate key cellular functions. Over half of theknown tyrosine kinases are implicated in human cancers and are thereforehighly promising drug targets. In one embodiment, the present inventioncontemplates targeting with drug combinations (given together orsequentially).

This invention is described in preferred embodiments in the followingdescription with reference to the Figures, in which like numbersrepresent the same or similar elements. Reference throughout thisspecification to “one embodiment,” “an embodiment,” or similar languagemeans that a particular feature, structure, or characteristic describedin connection with the embodiment is included in at least one embodimentof the present invention. Thus, appearances of the phrases “in oneembodiment,” “in an embodiment,” and similar language throughout thisspecification may, but do not necessarily, all refer to the sameembodiment.

In one embodiment, the invention relates to a method of treating cancer,comprising: a) providing i) a subject with cancer, ii) a inhibitor of atyrosine kinase, and iii) an EGFR inhibitor, and b) treating saidsubject with said inhibitors. In one embodiment, said cancer is breastcancer. In one embodiment, said breast cancer is HER2+ breast cancer. Inone embodiment, said tyrosine kinase is Bruton's Tyrosine Kinase. In oneembodiment, said tyrosine kinase is a variant of Bruton's TyrosineKinase comprising an amino-terminal extension. In one embodiment, saidEGFR inhibitor is lapatinib. In one embodiment, said EGFR inhibitor isselected from the group consisting of gefitinib, erlotinib, cetuximab,panitumumab, and vandetanib. In one embodiment, said tyrosine kinaseinhibitor is selected from the group consisting of ibrutinib(PCI-32765), AVL-292 and CGI-1746. In one embodiment, said treating saidsubject with said inhibitors is sequential. In one embodiment, saidtreating said subject with said inhibitors is simultaneous. In oneembodiment, treating with said inhibitors results in reducedproliferation of at least some of said cancer cells within said subject.

In one embodiment, the invention relates to a method of treating cancer,comprising: a) providing i) a subject with cancer and ii) AVL-292, iii)lapatinib, and b) treating said subject with said AVL-292 and lapatinib.In one embodiment, said treating said subject with said inhibitors issequential. In one embodiment, said treating said subject with saidinhibitors is simultaneous. In one embodiment, said cancer is breastcancer. In one embodiment, said breast cancer comprises Her-2 positivecells.

In one embodiment, the invention relates to a method of treating cancer,comprising: a) providing i) AVL-292 and ii) a subject with cancer,wherein said subject has been treated with lapatinib, and b) treatingsaid subject with said AVL-292. In one embodiment, said cancer is breastcancer. In one embodiment, said breast cancer comprises Her-2 positivecells.

In one embodiment, the invention relates to a method of treating cancer,comprising: a) providing i) ibrutinib and ii) a subject with cancer,wherein said subject has been treated with lapatinib, and b) treatingsaid subject with said ibrutinib. In one embodiment, said cancer isbreast cancer. In one embodiment, said breast cancer comprises Her-2positive cells.

In one embodiment, the invention relates to a pharmaceutical anticancercomposition comprising AVL-292 and lapatinib.

In one embodiment, the invention relates to a pharmaceutical anticancercomposition comprising ibrutinib and lapatinib.

The described features, structures, or characteristics of the inventionmay be combined in any suitable manner in one or more embodiments. Inthe following description, numerous specific details are recited toprovide a thorough understanding of embodiments of the invention. Oneskilled in the relevant art will recognize, however, that the inventionmay be practiced without one or more of the specific details, or withother methods, components, materials, and so forth. In other instances,well-known structures, materials, or operations are not shown ordescribed in detail to avoid obscuring aspects of the invention.

Definitions

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

The phrase “chosen from A, B, and C” as used herein, means selecting oneor more of A, B, C.

As used herein, absent an express indication to the contrary, the term“or” when used in the expression “A or B,” where A and B refer to acomposition, disease, product, etc., means one or the other, or both. Asused herein, the term “comprising” when placed before the recitation ofsteps in a method means that the method encompasses one or more stepsthat are additional to those expressly recited, and that the additionalone or more steps may be performed before, between, and/or after therecited steps. For example, a method comprising steps a, b, and cencompasses a method of steps a, b, x, and c, a method of steps a, b, c,and x, as well as method of steps x, a, b, and c. Furthermore, the term“comprising” when placed before the recitation of steps in a method doesnot (although it may) require sequential performance of the listedsteps, unless the context clearly dictates otherwise. For example, amethod comprising steps a, b, and c encompasses, for example, a methodof performing steps in the order of steps a, c, and b, the order ofsteps c, b, and a, and the order of steps c, a, and b, etc.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weights, reaction conditions,and so forth as used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersin the specification and claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and without limiting theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parametersdescribing the broad scope of the invention are approximations, thenumerical values in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains standarddeviations that necessarily result from the errors found in thenumerical value's testing measurements.

The term “not” when preceding, and made in reference to, anyparticularly named molecule (mRNA, etc.) or phenomenon (such asbiological activity, biochemical activity, etc.) means that only theparticularly named molecule or phenomenon is excluded.

The term “altering” and grammatical equivalents as used herein inreference to the level of any substance and/or phenomenon refers to anincrease and/or decrease in the quantity of the substance and/orphenomenon, regardless of whether the quantity is determinedobjectively, and/or subjectively.

The terms “increase,” “elevate,” “raise,” and grammatical equivalentswhen used in reference to the level of a substance and/or phenomenon ina first sample relative to a second sample, mean that the quantity ofthe substance and/or phenomenon in the first sample is higher than inthe second sample by any amount that is statistically significant usingany art-accepted statistical method of analysis. In one embodiment, theincrease may be determined subjectively, for example when a patientrefers to their subjective perception of disease symptoms, such as pain,clarity of vision, etc. In another embodiment, the quantity of thesubstance and/or phenomenon in the first sample is at least 10% greaterthan the quantity of the same substance and/or phenomenon in a secondsample. In another embodiment, the quantity of the substance and/orphenomenon in the first sample is at least 25% greater than the quantityof the same substance and/or phenomenon in a second sample. In yetanother embodiment, the quantity of the substance and/or phenomenon inthe first sample is at least 50% greater than the quantity of the samesubstance and/or phenomenon in a second sample. In a further embodiment,the quantity of the substance and/or phenomenon in the first sample isat least 75% greater than the quantity of the same substance and/orphenomenon in a second sample. In yet another embodiment, the quantityof the substance and/or phenomenon in the first sample is at least 90%greater than the quantity of the same substance and/or phenomenon in asecond sample. Alternatively, a difference may be expressed as an“n-fold” difference.

The terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,” andgrammatical equivalents when used in reference to the level of asubstance and/or phenomenon in a first sample relative to a secondsample, mean that the quantity of substance and/or phenomenon in thefirst sample is lower than in the second sample by any amount that isstatistically significant using any art-accepted statistical method ofanalysis. In one embodiment, the reduction may be determinedsubjectively, for example when a patient refers to their subjectiveperception of disease symptoms, such as pain, clarity of vision, etc. Inanother embodiment, the quantity of substance and/or phenomenon in thefirst sample is at least 10% lower than the quantity of the samesubstance and/or phenomenon in a second sample. In another embodiment,the quantity of the substance and/or phenomenon in the first sample isat least 25% lower than the quantity of the same substance and/orphenomenon in a second sample. In yet another embodiment, the quantityof the substance and/or phenomenon in the first sample is at least 50%lower than the quantity of the same substance and/or phenomenon in asecond sample. In a further embodiment, the quantity of the substanceand/or phenomenon in the first sample is at least 75% lower than thequantity of the same substance and/or phenomenon in a second sample. Inyet another embodiment, the quantity of the substance and/or phenomenonin the first sample is at least 90% lower than the quantity of the samesubstance and/or phenomenon in a second sample. Alternatively, adifference may be expressed as an “n-fold” difference.

A number of terms herein relate to cancer. “Cancer” is intended hereinto encompass all forms of abnormal or improperly regulated reproductionof cells in a subject. “Subject” and “patient” are used hereininterchangeably, and a subject may be any mammal but is preferably ahuman. A “reference subject” herein refers to an individual who does nothave cancer. The “reference subject” thereby provides a basis to whichanother cell (for example a cancer cell) can be compared.

The growth of cancer cells (“growth” herein referring generally to celldivision but also to the growth in size of masses of cells) ischaracteristically uncontrolled or inadequately controlled, as is thedeath (“apoptosis”) of such cells. Local accumulations of such cellsresult in a tumor. More broadly, and still denoting “tumors” herein areaccumulations ranging from a cluster of lymphocytes at a site ofinfection to vascularized overgrowths, both benign and malignant. A“malignant” tumor (as opposed to a “benign” tumor) herein comprisescells that ten to migrate to nearby tissues, including cells that maytravel through the circulatory system to invade or colonize tissues ororgans at considerable remove from their site of origin in the “primarytumor,” so-called herein. Metastatic cells are adapted to penetrateblood vessel wells to enter (“intravasate”) and exit (“extravasate”)blood vessels. Tumors capable of releasing such cells are also referredto herein as “metastatic.” The term is used herein also to denote anycell in such a tumor that is capable of such travel, or that is enroute, or that has established a foothold in a target tissue. Forexample, a metastatic breast cancer cell that has taken root in the lungis referred to herein as a “lung metastasis.” Metastatic cells may beidentified herein by their respective sites of origin and destination,such as “breast-to-bone metastatic.” In the target tissue, a colony ofmetastatic cells can grow into a “secondary tumor,” so called herein.

Primary tumors are thought to derive from a benign or normal cellthrough a process referred to herein as “cancer progression.” Accordingto this view, the transformation of a normal cell to a cancer cellrequires changes (usually many of them) in the cell's biochemistry. Thechanges are reflected clinically as the disease progresses throughstages. Even if a tumor is “clonogenic” (as used herein, an accumulationof the direct descendants of a parent cell), the biochemistry of theaccumulating cells changes in successive generations, both because theexpression of the genes (controlled by so-called “epigenetic” systems)of these cells becomes unstable and because the genomes themselveschange. In normal somatic cells, the genome (that is, all the genes ofan individual) is stored in the chromosomes of each cell (setting asidethe mitochondrial genome). The number of copies of any particular geneis largely invariant from cell to cell. By contrast, “genomicinstability” is characteristic of cancer progression. A genome in acancer cell can gain (“genomic gain”) or lose (“genomic loss”) genes,typically because an extra copy of an entire chromosome appears(“trisomy”) or a region of a chromosome replicates itself (“genomicgain” or, in some cases, “genomic amplification”) or drops out when thecell divides. Thus, the “copy number” of a gene or a set of genes,largely invariant among normal cells, is likely to change in cancercells (referred to herein as a “genomic event”), which affects the totalexpression of the gene or gene set and the biological behavior(“phenotype”) of descendent cells. Thus, in cancer cells, “geneactivity” herein is determined not only by the multiple “layers” ofepigenetic control systems and signals that call forth expression of thegene but by the number of times that gene appears in the genome. Theterm “epigenetic” herein refers to any process in an individual that, inoperation, affects the expression of a gene or a set of genes in thatindividual, and stands in contrast to the “genetic” processes thatgovern the inheritance of genes in successive generations of cells orindividuals.

Certain regions of chromosomes, depending upon the specific type ofcancer, have proven to be hot spots for genomic gain inasmuch asincreases in copy number in the genomes of cells from multiple donorstend to occur in one or a few specific regions of a specific chromosome.Such hot spots are referred to herein as sites of “recurrent genomicgain.” The term is to be distinguished from “recurrent cancer,” whichrefers to types of cancer that are likely to recur after an initialcourse of therapy, resulting in a “relapse.” A number of terms hereinrelate to methods that enable the practitioner to examine many distinctgenes at once. By these methods, sets of genes (“gene sets”) have beenidentified wherein each set has biologically relevant and distinctiveproperties as a set. Devices (which may be referred to herein as“platforms”) in which each gene in a significant part of an entiregenome is isolated and arranged in an array of spots, each spot havingits own “address,” enable one to detect, quantitatively, many thousandsof the genes in a cell. More precisely, these “microarrays” typicallydetect expressed genes (an “expressed” gene is one that is activelytransmitting its unique biochemical signal to the cell in which the generesides). Microarray data, inasmuch as they display the expression ofmany genes at once, permit the practitioner to view “gene expressionprofiles” in a cell and to compare those profiles cell-to-cell toperform so-called “comparative analyses of expression profiles.” Suchmicroarray-based “expression data” are capable of identifying genes thatare “over-expressed” (or under-expressed) in, for example, a diseasecondition. An over-expressed gene may be referred to herein as having ahigh “expression score.”

The aforementioned methods for examining gene sets employ a number ofwell-known methods in molecular biology, to which references are madeherein. A gene is a heritable chemical code resident in, for example, acell, vims, or bacteriophage that an organism reads (decodes, decrypts,transcribes) as a template for ordering the structures of biomoleculesthat an organism synthesizes to impart regulated function to theorganism. Chemically, a gene is a heteropolymer comprised of subunits(“nucleotides”) arranged in a specific sequence. In cells, suchheteropolymers are deoxynucleic acids (“DNA”) or ribonucleic acids(“RNA”). DNA forms long strands. Characteristically, these strands occurin pairs. The first member of a pair is not identical in nucleotidesequence to the second strand, but complementary. The tendency of afirst strand to bind in this way to a complementary second strand (thetwo strands are said to “anneal” or “hybridize”), together with thetendency of individual nucleotides to line up against a single strand ina complementarily ordered manner accounts for the replication of DNA.

Experimentally, nucleotide sequences selected for their complementaritycan be made to anneal to a strand of DNA containing one or more genes. Asingle such sequence can be employed to identify the presence of aparticular gene by attaching itself to the gene. This so called “probe”sequence is adapted to carry with it a “marker” that the investigatorcan readily detect as evidence that the probe struck a target. As usedherein, the term “marker” relates to any surrogate the artisan may useto “observe” an event or condition that is difficult or impossible todetect directly. In some contexts herein, the marker is said to “target”the condition or event. In other contexts, the condition or event isreferred to as the target for the marker. Sequences used as probes maybe quite small (e.g., “oligonucleotides” of <20 nucleotides) or quitelarge (e.g., a sequence of 100,000 nucleotides in DNA from a “bacterialartificial chromosome” or “BAC”).

A BAC is a bacterial chromosome (or a portion thereof) with a “foreign”(typically, human) DNA fragment inserted in it. BACs are employed in atechnique referred to herein as “fluorescence in situ hybridization” or“FISH.” A BAC or a portion of a BAC is constructed that has (1) asequence complementary to a region of interest on a chromosome and (2) amarker whose presence is discernible by fluorescence. The chromosomes ofa cell or a tissue are isolated (on a glass slide, for example) andtreated with the BAC construct. Excess construct is washed away and thechromosomes examined microscopically to find chromosomes or, moreparticularly, identifiable regions of chromosomes that fluoresce.

Alternatively, such sequences can be delivered in pairs selected tohybridize with two specific sequences that bracket a gene sequence. Acomplementary strand of DNA then forms between the “primer pair.” In onewell-known method, the “polymerase chain reaction” or “PCR,” theformation of complementary strands can be made to occur repeatedly in anexponential amplification. A specific nucleotide sequence so amplifiedis referred to herein as the “amplicon” of that sequence. “QuantitativePCR” or “qPCR” herein refers to a version of the method that allows theartisan not only to detect the presence of a specific nucleic acidsequence but also to quantify how many copies of the sequence arepresent in a sample, at least relative to a control. As used herein,“qRTPCR” may refer to “quantitative real-time PCR,” used interchangeablywith “qPCR” as a technique for quantifying the amount of a specific DNAsequence in a sample. However, if the context so admits, the sameabbreviation may refer to “quantitative reverse transcriptase PCR,” amethod for determining the amount of messenger RNA present in a sample.Since the presence of a particular messenger RNA in a cell indicatesthat a specific gene is currently active (being expressed) in the cell,this quantitative technique finds use, for example, in gauging the levelof expression of a gene.

Collectively, the genes of an organism constitute its genome. The term“genomic DNA” may refer herein to the entirety of an organism's DNA orto the entirety of the nucleotides comprising a single gene in anorganism. A gene typically contains sequences of nucleotides devoted tocoding (“exons”), and non-coding sequences that contribute in one way oranother to the decoding process (“introns”).

The term “gene” refers to a nucleic acid (e.g., DNA) comprisingcovalently linked nucleotide monomers arranged in a particular sequencethat comprises a coding sequence necessary for the production of apolypeptide or precursor or RNA (e.g., tRNA, siRNA, rRNA, etc.). Thepolypeptide can be encoded by a full-length coding sequence or by anyportion of the coding sequence so long as the desired activities orfunctional properties (e.g., enzymatic activity, ligand binding, signaltransduction, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region together with the sequenceslocated adjacent to the coding region on both the 5′ and 3′ ends, suchthat the gene corresponds to the length of the full-length mRNA (alsoreferred to as “pre-mRNA,” “nuclear RNA,” or “primary transcript RNA”)transcribed from it. The sequences that are located 5′ of the codingregion and are present on the mRNA are referred to as 5′ untranslatedsequences. The sequences that are located 3′ or downstream of the codingregion and that are present on the mRNA are referred to as 3′untranslated sequences. The term “gene” encompasses both cDNA (thecoding region(s) only) and genomic forms of a gene. A genomic form orclone of a gene contains the coding region, which may be interruptedwith non-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are removed or “spliced out” from thenuclear or primary transcript, and are therefore absent in the messengerRNA (mRNA) transcript. The mRNA functions during translation to specifythe sequence or order of amino acids in a nascent polypeptide.

Encoding in DNA (and messenger RNA) is accomplished by 3-memberednucleotide sequences called “codons.” Each codon encrypts an amino acid,and the sequence of codons encrypts the sequence of amino acids thatidentifies a particular protein. The code for a given gene is embeddedin a (usually) much longer nucleotide sequence and is distinguishable tothe cell's decoding system from the longer sequence by a “start codon”and a “stop” codon. The decoding system reads the sequence framed bythese two codons (the so-called “open reading frame”). The readable codeis transcribed into messenger RNA which itself comprises sites thatensure coherent translation of the code from nucleic acid to protein. Inparticular, the open reading frame is delimited by a so-called“translation initiation” codon and “translation termination” codon.

The term “plasmid” as used herein, refers to a small, independentlyreplicating, piece of DNA. Similarly, the term “naked plasmid” refers toplasmid DNA devoid of extraneous material typically used to effecttransfection. As used herein, a “naked plasmid” refers to a plasmidsubstantially free of calcium-phosphate, DEAE-dextran, liposomes, and/orpolyamines. As used herein, the term “purified” refers to molecules(polynucleotides or polypeptides) that are removed from their naturalenvironment, isolated or separated. “Purified” molecules are at least50% free, preferably at least 75% free, and more preferably at least 90%free from other components with which they are naturally associated.

The term “recombinant DNA” refers to a DNA molecule that is comprised ofsegments of DNA joined together by means of molecular biologytechniques. Similarly, the term “recombinant protein” refers to aprotein molecule that is expressed from recombinant DNA.

The term “fusion protein” as used herein refers to a protein formed byexpression of a hybrid gene made by combining two gene sequences.Typically this is accomplished by cloning a cDNA into an expressionvector in frame (i.e., in an arrangement that the cell can transcribe asa single mRNA molecule) with an existing gene. The fusion partner mayact as a reporter (e.g., βgal) or may provide a tool for isolationpurposes (e.g., GST).

Where an amino acid sequence is recited herein to refer to an amino acidsequence of a protein molecule, “amino acid sequence” and like terms,such as “polypeptide” or “protein” are not meant to limit the amino acidsequence to the complete, native amino acid sequence associated with therecited protein molecule. Rather the terms “amino acid sequence” and“protein” encompass partial sequences, and modified sequences.

The term “wild type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild type gene is the variant mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene.

In contrast, the terms “modified,” “mutant,” and “variant” (when thecontext so admits) refer to a gene or gene product that displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Insome embodiments, the modification comprises at least one nucleotideinsertion, deletion, or substitution.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid and is referred to using the functional term “substantiallyhomologous.” The term “inhibition of binding,” when used in reference tonucleic acid binding, refers to reduction in binding caused bycompetition of homologous sequences for binding to a target sequence.The inhibition of hybridization of the completely complementary sequenceto the target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely homologous sequence to a target under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target that lacks even a partial degreeof complementarity (e.g., less than about 30% identity); in the absenceof non-specific binding the probe will not hybridize to the secondnon-complementary target. When used in reference to a single-strandednucleic acid sequence, the term “substantially homologous” refers to anyprobe that can hybridize (i.e., it is the complement of) thesingle-stranded nucleic acid sequence under conditions of low stringencyas described above.

As used herein, the term “competes for binding” when used in referenceto a first and a second polypeptide means that the first polypeptidewith an activity binds to the same substrate as does the secondpolypeptide with an activity. In one embodiment, the second polypeptideis a variant of the first polypeptide (e.g., encoded by a differentallele) or a related (e.g., encoded by a homolog) or dissimilar (e.g.,encoded by a second gene having no apparent relationship to the firstgene) polypeptide. The efficiency (e.g., kinetics or thermodynamics) ofbinding by the first polypeptide may be the same as or greater than orless than the efficiency of substrate binding by the second polypeptide.For example, the equilibrium binding constant (Ko) for binding to thesubstrate may be different for the two polypeptides.

As used herein, the term “hybridization” refers to the pairing ofcomplementary nucleic acids. Hybridization and the strength ofhybridization (i.e., the strength of the association between the nucleicacids) is impacted by such factors as the degree of complementaritybetween the nucleic acids, stringency of the conditions involved, theT_(m) of the formed hybrid, and the G:C ratio within the nucleic acids.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985] [7]). Otherreferences include more sophisticated computations that take structuralas well as sequence characteristics into account for the calculation ofT_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Those skilled in the art will recognizethat “stringency” conditions may be altered by varying the parametersjust described either individually or in concert. With “high stringency”conditions, nucleic acid base pairing will occur only between nucleicacid fragments that have a high frequency of complementary basesequences (e.g., hybridization under “high stringency” conditions mayoccur between homologs with 85-100% identity, preferably 70-100%identity). With medium stringency conditions, nucleic acid base pairingwill occur between nucleic acids with an intermediate frequency ofcomplementary base sequences (e.g., hybridization under “mediumstringency” conditions may occur between homologs with 50-70% identity).Thus, conditions of “weak” or “low” stringency are often required withnucleic acids that are derived from organisms that are geneticallydiverse, as the frequency of complementary sequences is usually less.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution comprising 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 100 to about 1000 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution comprising 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42° C. when aprobe of about 100 to about 1000 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42° C. in a solution comprising 5×SSPE (43.8 g/l NaCl,6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH),0.1% SDS, 5×Denhardt's reagent [50×Denhardt's contains per 500 ml: 5 gFicoll® (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 g/mldenatured salmon sperm DNA followed by washing in a solution comprising5×SSPE, 0.1% SDS at 42° C. when a probe of about 100 to about 1000nucleotides in length is employed.

The term “equivalent” when made in reference to a hybridizationcondition as it relates to a hybridization condition of interest meansthat the hybridization condition and the hybridization condition ofinterest result in hybridization of nucleic acid sequences which havethe same range of percent (%) homology. For example, if a hybridizationcondition of interest results in hybridization of a first nucleic acidsequence with other nucleic acid sequences that have from 85% to 95%homology to the first nucleic acid sequence, then another hybridizationcondition is said to be equivalent to the hybridization condition ofinterest if this other hybridization condition also results inhybridization of the first nucleic acid sequence with the other nucleicacid sequences that have from 85% to 95% homology to the first nucleicacid sequence.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “sequenceidentity”, “percentage of sequence identity”, and “substantialidentity”. A “reference sequence” is a defined sequence used as a basisfor a sequence comparison; a reference sequence may be a subset of alarger sequence, for example, as a segment of a full-length cDNAsequence given in a sequence listing or may comprise a complete genesequence. Generally, a reference sequence is at least 20 nucleotides inlength, frequently at least 25 nucleotides in length, and often at least50 nucleotides in length. Since two polynucleotides may each (1)comprise a sequence (i.e., a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) mayfurther comprise a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window”, as usedherein, refers to a conceptual segment of at least 20 contiguousnucleotide positions wherein a polynucleotide sequence may be comparedto a reference sequence of at least 20 contiguous nucleotides andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman (Smithand Waterman, Adv. Appl. Math., 2: 482, 1981 [8]) by the homologyalignment algorithm of Needleman and Wunsch (Needleman and Wunsch, IMol. Biol. 48:443, 1970 [9]), by the search for similarity method ofPearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci., U.S.A.,85:2444, 1988 [10]), by computerized implementations of these algorithms(GAP, BESTFIT, PASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, Genetics Computer Group, Madison, Wis.), or byinspection, and the best alignment (i.e., resulting in the highestpercentage of homology over the comparison window) generated by thevarious methods is selected. The term “sequence identity” means that twopolynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. The terms “substantial identity” as used herein denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 85 percent sequence identity,preferably at least 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 nucleotide positions, frequentlyover a window of at least 25-50 nucleotides, wherein the percentage ofsequence identity is calculated by comparing the reference sequence tothe polynucleotide sequence which may include deletions or additionswhich total 20 percent or less of the reference sequence over the windowof comparison. The reference sequence may be a subset of a largersequence, for example, as a segment of the full-length sequences of thecompositions claimed in the present invention.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having acidic side chains is glutamic acid and asparticacid; a group of amino acids having basic side chains is lysine,arginine, and histidine; and a group of amino acids havingsulfur-containing side chains is cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

“Amplification” is used herein in two different ways. A given genetypically appears in a genome once, on one chromosome. Since chromosomesin somatic cells of eukaryotes are in general paired, two copies oralleles of each gene are found. In some conditions, such as cancer,replication of chromosome pairs during cell division is disturbed sothat multiple copies of a gene or chromosome accrue over successivegenerations. The phenomenon is referred to generally (and herein) as“amplification.”

In the context of molecular biological experimentation, the term is useddifferently. Experimentally, “amplification” is used in relation to aspecial case of nucleic acid replication involving template specificity.It is to be contrasted with non-specific template replication (i.e.,replication that is template-dependent but not dependent on a specifictemplate). Template specificity is here distinguished from fidelity ofreplication (i.e., synthesis of the proper polynucleotide sequence) andnucleotide (ribo- or deoxyribo-) specificity. Template specificity isfrequently described in terms of “target” specificity. Target sequencesare “targets” in the sense that they are sought to be sorted out fromother nucleic acid. Amplification techniques have been designedprimarily for this sorting out.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, under theconditions in which they are used, will process only specific sequencesof nucleic acids in a heterogeneous mixture of nucleic acids. Inparticular, Taq and Pfu polymerases, by virtue of their ability tofunction at high temperature, are found to display high specificity forthe sequences bounded and thus defined by the primers; the hightemperature results in thermodynamic conditions that favor primerhybridization with the target sequences and not hybridization withnon-target sequences.

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of “target”(defined below). In contrast, “background template” is used in referenceto nucleic acid other than sample template that may or may not bepresent in a sample. Background template is most often inadvertent. Itmay be the result of carryover, or it may be due to the presence ofnucleic acid contaminants sought to be purified away from the sample.For example, nucleic acids from organisms other than those to bedetected may be present as background in a test sample.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particularsequences. It is contemplated that any probe used in the presentinvention will be labeled with any “reporter molecule,” so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label.

As used herein, the term “target,” when used in reference to thepolymerase chain reaction, refers to the region of nucleic acid boundedby the primers used for polymerase chain reaction. Thus, the “target” issought to be sorted out from other nucleic acid sequences. A “segment”is defined as a region of nucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of Mullis (U.S. Pat. No. 4,683,195 [11], U.S. Pat. No.4,683,202 [12], and U.S. Pat. No. 4,965,188 [13], hereby incorporated byreference), that describe a method for increasing the concentration of asegment of a target sequence in a mixture of genomic DNA without cloningor purification. This process for amplifying the target sequenceconsists of introducing a large excess of two oligonucleotide primers tothe DNA mixture containing the desired target sequence, followed by aprecise sequence of thermal cycling in the presence of a DNA polymerase.The two primers are complementary to their respective strands of thedouble stranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing, and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one “cycle”; there can be numerous “cycles”) toobtain a high concentration of an amplified segment of the desiredtarget sequence. The length of the amplified segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to as the “polymerase chain reaction” (hereinafter “PCR”).Because the desired amplified segments of the target sequence become thepredominant sequences (in terms of concentration) in the mixture, theyare said to be “PCR amplified.”

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant nucleic acid with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is present in a form or settingthat is different from that in which it is found in nature. In contrast,non-isolated nucleic acids are nucleic acids such as DNA and RNA foundin the state they exist in nature. For example, a given DNA sequence(e.g., a gene) is found on the host cell chromosome in proximity toneighboring genes; RNA sequences, such as a specific mRNA sequenceencoding a specific protein, are found in the cell as a mixture withnumerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding gene includes, by way of example, suchnucleic acid in cells ordinarily expressing gene where the nucleic acidis in a chromosomal location different from that of natural cells, or isotherwise flanked by a different nucleic acid sequence than that foundin nature. The isolated nucleic acid, oligonucleotide, or polynucleotidemay be present in single-stranded or double-stranded form. When anisolated nucleic acid, oligonucleotide or polynucleotide is to beutilized to express a protein, the oligonucleotide or polynucleotidewill contain at a minimum the sense or coding strand (i.e., theoligonucleotide or polynucleotide may single-stranded), but may containboth the sense and anti-sense strands (i.e., the oligonucleotide orpolynucleotide may be double-stranded).

The terms “fragment” and “portion” when used in reference to anucleotide sequence (as in “a portion of a given nucleotide sequence”)refers to partial segments of that sequence. The fragments may range insize from four nucleotides to the entire nucleotide sequence minus onenucleotide (10 nucleotides, 20, 30, 40, 50, 100, 200, etc.).

Similarly, the terms “fragment” and “portion” when used in reference toa polypeptide sequence refers to partial segments of that sequence. Insome embodiments, the portion has an amino-terminal and/orcarboxy-terminal deletion as compared to the native protein, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the amino acid sequence deduced from a full-length cDNAsequence. Fragments are preferably at least 4 amino acids long, morepreferably at least 50 amino acids long, and most preferably at least 50amino acids long or longer (the entire amino acid sequence minus onamino acid). In particularly preferred embodiments, the portioncomprises the amino acid residues required for intermolecular binding ofthe compositions of the present invention with its various ligandsand/or substrates.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four consecutive amino acid residues tothe entire amino acid sequence minus one amino acid.

As used herein the term “coding region” when used in reference tostructural gene refers to the nucleotide sequences that encode the aminoacids found in the nascent polypeptide as a result of translation of amRNA molecule. The coding region is bounded, in eukaryotes, on the 5′side by the nucleotide triplet “ATG” that encodes the initiatormethionine and on the 3′ side by one of the three triplets which specifystop codons (i.e., TAA, TAG, TGA).

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques. Similarly, the term “recombinantprotein” or “recombinant polypeptide” as used herein refers to a proteinmolecule that is expressed from a recombinant DNA molecule.

The term “native protein” as used herein to indicate that a protein doesnot contain amino acid residues encoded by vector sequences, that arethe native protein contains only those amino acids found in the proteinas it occurs in nature. A native protein may be produced by recombinantmeans or may be isolated from a naturally occurring source.

The term “Southern blot,” refers to the analysis of DNA on agarose oracrylamide gels to fractionate the DNA according to size followed bytransfer of the DNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized DNA is then probedwith a labeled probe to detect DNA species complementary to the probeused. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists(Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, NY, pp. 9.31-9.58, 1989 [14]).

The term “Northern blot,” as used herein refers to the analysis of RNAby electrophoresis of RNA on agarose gels to fractionate the RNAaccording to size followed by transfer of the RNA from the gel to asolid support, such as nitrocellulose or a nylon membrane. Theimmobilized RNA is then probed with a labeled probe to detect RNAspecies complementary to the probe used. Northern blots are a standardtool of molecular biologists (Sambrook, et al., supra, pp. 7.39-7.52,1989 [15]).

The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of radiolabelled antibodies

As used herein, the term “transgenic” refers to a cell or organism whosegenome has been heritably altered by genetically engineering into thegenome a gene (“transgene”) not normally part of it or removing from ita gene ordinarily present (a “knockout” gene). The “transgene” or“foreign gene” may be placed into an organism by introducing it intonewly fertilized eggs or early embryos. The term “foreign gene” refersto any nucleic acid (e.g., gene sequence) that is introduced into thegenome of an animal by experimental manipulations and may include genesequences found in that animal so long as the introduced gene does notreside in the same location as does the naturally-occurring gene.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

As used herein, the term host cell refers to any eukaryotic orprokaryotic cell (e.g. bacterial cells such as E. coli, yeast cells,mammalian cells, avian cells, amphibian cells, plant cells, fish cells,and insect cells), whether located in vitro or in vivo. For example,host cells may be located in a transgenic animal.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell in the sense thatthe foreign DNA will be passed on to daughter cells. The termencompasses transfections of foreign DNA into the cytoplasm only. Ingeneral, however, the foreign DNA reaches the nucleus of the transfectedcell and persists there for several days. During this time the foreignDNA is subject to the regulatory controls that govern the expression ofendogenous genes in the chromosomes. The term “transient transfectant”refers to cells that have taken up foreign DNA but have failed tointegrate this DNA. The term “transient transfection” encompassestransfection of foreign DNA into the cytoplasm only.

The term “calcium phosphate co-precipitation” refers to a technique forthe introduction of nucleic acids into a cell. The uptake of nucleicacids by cells is enhanced when the nucleic acid is presented as acalcium phosphate-nucleic acid co-precipitate. The original technique ofis modified to optimize conditions for particular types of cells. Theart is well aware of these numerous modifications.

A “composition comprising a given polynucleotide sequence” as usedherein refers broadly to any composition containing the givenpolynucleotide sequence. Such compositions may be employed ashybridization probes, typically in an aqueous solution containing salts(e.g., NaCl), detergents (e.g., SDS), and other components (e.g.,Denhardt's solution, dry milk, salmon sperm DNA, etc.).

The terms “N-terminus” “NH₂-terminus” and “amino-terminus” refer to theamino acid residue corresponding to the methionine encoded by the startcodon (e.g., position or residue 1). In contrast the terms “C-terminus”“COOR-terminus” and “carboxy terminus” refer to the amino acid residueencoded by the final codon (e.g., last or final residue prior to thestop codon). The term “conservative substitution” as used herein refersto a change that takes place within a family of amino acids that arerelated in their side chains. Genetically encoded amino acids can bedivided into four families: (1) acidic (aspartate, glutamate); (2) basic(lysine, arginine, histidine): nonpolar (alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); and (4)uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3)aliphatic (glycine, alanine, valine, leucine, isoleucine, serine,threonine), with serine and threonine optionally be grouped separatelyas aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine). Whether a change in theamino acid sequence of a peptide results in a functional homolog can bereadily determined by assessing the ability of the variant peptide tofunction in a fashion similar to the wild-type protein. Peptides havingmore than one replacement can readily be tested in the same manner. Incontrast, the term “non-conservative substitution” refers to a change inwhich an amino acid from one family is replaced with an amino acid fromanother family (e.g., replacement of a glycine with a tryptophan).Guidance in determining which amino acid residues can be substituted,inserted, or deleted without abolishing biological activity can be foundusing computer programs (e.g., LASERGENE software, DNASTAR Inc.,Madison, Ws.

A peptide sequence and nucleotide sequence may be “endogenous” or“heterologous” (i.e., “foreign”). The term “endogenous” refers to asequence which is naturally found in the cell or virus into which it isintroduced so long as it does not contain some modification relative tothe naturally-occurring sequence. The term “heterologous” refers to asequence which is not endogenous to the cell or virus into which it isintroduced. For example, heterologous DNA includes a nucleotide sequencewhich is ligated to, or is manipulated to become ligated to, a nucleicacid sequence to which it is not ligated in nature, or to which it isligated at a different location in nature. Heterologous DNA alsoincludes a nucleotide sequence which is naturally found in the cell orvirus into which it is introduced and which contains some modificationrelative to the naturally-occurring sequence. Generally, although notnecessarily, heterologous DNA encodes heterologous RNA and heterologousproteins that are not normally produced by the cell or virus into whichit is introduced. Examples of heterologous DNA include reporter genes,transcriptional and translational regulatory sequences, DNA sequenceswhich encode selectable marker proteins (e.g., proteins which conferdrug resistance), etc. In preferred embodiments, the terms “heterologousantigen” and “heterologous sequence” refer to a non-hepadna virusantigen or amino acid sequence including but not limited to microbialantigens, mammalian antigens and allergen antigens.

The terms “peptide,” “peptide sequence,” “amino acid sequence,”“polypeptide,” and “polypeptide sequence” are used interchangeablyherein to refer to at least two amino acids or amino acid analogs whichare covalently linked by a peptide bond or an analog of a peptide bond.The term peptide includes oligomers and polymers of amino acids or aminoacid analogs. The term peptide also includes molecules which arecommonly referred to as peptides, which generally contain from about two(2) to about twenty (20) amino acids. The term peptide also includesmolecules which are commonly referred to as polypeptides, whichgenerally contain from about twenty (20) to about fifty amino acids(50). The term peptide also includes molecules which are commonlyreferred to as proteins, which generally contain from about fifty (50)to about three thousand (3000) amino acids. The amino acids of thepeptide may be L-amino acids or D-amino acids. A peptide, polypeptide orprotein may be synthetic, recombinant or naturally occurring. Asynthetic peptide is a peptide which is produced by artificial means invitro.

The terms “oligosaccharide” and “OS” antigen refer to a carbohydratecomprising up to ten component sugars, either 0 or N linked to the nextsugar. Likewise, the terms “polysaccharide” and “PS” antigen refer topolymers of more than ten monosaccharide residues linked glycosidicallyin branched or unbranched chains

As used herein, the term “mammalian sequence” refers to synthetic,recombinant or purified sequences (preferably sequence fragmentscomprising at least one B cell epitope) of a mammal. Exemplary mammaliansequences include cytokine sequence, MHC class I heavy chain sequences,MHC class II alpha and beta chain sequences, and amyloid 13-peptidesequences.

The terms “mammals” and “mammalian” refer animals of the class mammaliawhich nourish their young by fluid secreted from mammary glands of themother, including human beings. The class “mammalian” includes placentalanimals, marsupial animals, and monotrematal animals. An exemplary“mammal” may be a rodent, primate (including simian and human) ovine,bovine, ruminant, lagomorph, porcine, caprine, equine, canine, feline,ave, etc. Preferred non-human animals are selected from the orderRodentia.

Preferred embodiments of the present invention are primarily directed tovertebrate (backbone or notochord) members of the animal kingdom.

The terms “patient” and “subject” refer to a mammal that may be treatedusing the methods of the present invention.

The term “control” refers to subjects or samples which provide a basisfor comparison for experimental subjects or samples. For instance, theuse of control subjects or samples permits determinations to be maderegarding the efficacy of experimental procedures. In some embodiments,the term “control subject” refers to a subject that which receives amock treatment (e.g., saline alone).

The terms diluent and “diluting agent” as used herein refer to agentsused to diminish the strength of an admixture. Exemplary diluentsinclude water, physiological saline solution, human serum albumin, oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents, antibacterial agents such as benzyl alcohol, antioxidants suchas ascorbic acid or sodium bisulphite, chelating agents such as ethylenediamine-tetra-acetic acid, buffers such as acetates, citrates orphosphates and agents for adjusting the osmolarity, such as sodiumchloride or dextrose.

The terms “carrier” and “vehicle” as used herein refer to usuallyinactive accessory substances into which a pharmaceutical substance issuspended. Exemplary carriers include liquid carriers (such as water,saline, culture medium, saline, aqueous dextrose, and glycols) and solidcarriers (such as carbohydrates exemplified by starch, glucose, lactose,sucrose, and dextrans, anti-oxidants exemplified by ascorbic acid andglutathione, and hydrolyzed proteins.

The term “derived” when in reference to a peptide derived from a source(such as a microbe, cell, etc.) as used herein is intended to refer to apeptide which has been obtained (e.g., isolated, purified, etc.) fromthe source. Alternatively, or in addition, the peptide may begenetically engineered and/or chemically synthesized.

The terms “operably linked,” “in operable combination” and “in operableorder” as used herein refer to the linkage of nucleic acid sequencessuch that they perform their intended function. For example, operablylinking a promoter sequence to a nucleotide sequence of interest refersto linking the promoter sequence and the nucleotide sequence of interestin a manner such that the promoter sequence is capable of directing thetranscription of the nucleotide sequence of interest and/or thesynthesis of a polypeptide encoded by the nucleotide sequence ofinterest. Similarly, operably linking a nucleic acid sequence encoding aprotein of interest means linking the nucleic acid sequence toregulatory and other sequences in a manner such that the protein ofinterest is expressed. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

The terms “C-terminal portion,” “COOR-terminal portion,” “carboxyterminal portion,” “C-terminal domain,” “COOR-terminal domain,” and“carboxy terminal domain,” when used in reference to an amino acidsequence of interest refer to the amino acid sequence (and portionsthereof that is located from approximately the middle of the amino acidsequence of interest to the C-terminal-most amino acid residue of thesequence of interest. The terms “specific binding,” “bindingspecificity,” and grammatical equivalents thereof when made in referenceto the binding of a first molecule (such as a polypeptide, glycoprotein,nucleic acid sequence, etc.) to a second molecule (such as apolypeptide, glycoprotein, nucleic acid sequence, etc.) refer to thepreferential interaction between the first molecule with the secondmolecule as compared to the interaction between the second molecule witha third molecule. Specific binding is a relative term that does notrequire absolute specificity of binding; in other words, the term“specific binding” does not require that the second molecule interactwith the first molecule in the absence of an interaction between thesecond molecule and the third molecule. Rather, it is sufficient thatthe level of interaction between the first molecule and the secondmolecule is higher than the level of interaction between the secondmolecule with the third molecule. “Specific binding” of a first moleculewith a second molecule also means that the interaction between the firstmolecule and the second molecule is dependent upon the presence of aparticular structure on or within the first molecule; in other words thesecond molecule is recognizing and binding to a specific structure on orwithin the first molecule rather than to nucleic acids or to moleculesin general. For example, if a second molecule is specific for structure“A” that is on or within a first molecule, the presence of a thirdnucleic acid sequence containing structure A will reduce the amount ofthe second molecule which is bound to the first molecule.

For example, the term “has the biological activity of a specificallynamed protein” when made in reference to the biological activity of avariant of the specifically named protein refers, for example, to aquantity of binding of an antibody that is specific for the specificallynamed protein to the variant which is preferably greater than 50%(preferably from 50% to 500%, more preferably from 50% to 200%, mostpreferably from 50% to 100%), as compared to the quantity of binding ofthe same antibody to the specifically named protein.

Reference herein to any specifically named nucleotide sequence includeswithin its scope fragments, homologs, and sequences that hybridize understringent condition to the specifically named nucleotide sequence. Theterm “homolog” of a specifically named nucleotide sequence refers to anoligonucleotide sequence which exhibits greater than or equal to 50%identity to the sequence of interest. Alternatively, or in addition, ahomolog of any specifically named nucleotide sequence is defined as anoligonucleotide sequence which has at least 95% identity with thesequence of the nucleotide sequence in issue. In another embodiment, thesequence of the homolog has at least 90% identity, and preferably atleast 85% identity with the sequence of the nucleotide sequence inissue.

Exons, introns, genes and entire gene-sets are characteristicallylocatable with respect to one another. That is, they have generallyinvariant “genomic loci” or “genomic positions.” Genes distributedacross one or several chromosomes can be mapped to specific locations onspecific chromosomes. The field of “cytogenetics” addresses severalaspects of gene mapping. First, optical microscopy reveals features ofchromosomes that are useful as addresses for genes. In humans,chromosomes are morphologically distinguishable from one another andeach (except for the Y-chromosome) has two distinct arms separated by a“centromere.” Each arm has distinctive “bands” occupied by specificgenes. Disease-related changes in chromosome number and changes inbanding form the basis for diagnosing a number of diseases.“Microdissection” of chromosomes and DNA analysis of the microdissectedfragments have connected specific DNA sequences to specific locations onchromosomes. In cancer, a region of a chromosome may duplicate oramplify itself or drop out entirely. FISH, mentioned above, and“comparative genomic hybridization” (“CGH”) have extended the reach ofcytogenetic analysis to the extent of measuring genome alterationswithin and between individuals. CGH, for example, in which chromosomesfrom a normal cell are hybridized with a corresponding preparation froma cancer cell provides a means of directly determining cancer-relateddifferences in copy number of chromosomal regions.

“Targeted therapeutics” is used herein to denote any therapeuticmodality that affects only or primarily only the cells or tissuesselected (“targeted”) for treatment. A monoclonal antibody specific foran antigen expressed only by a target (if retained by the target) ishighly useful in targeted therapeutics. In the case of unwanted cellssuch as cancer cells, if the antibody doesn't induce destruction of thetarget directly, it may do so indirectly by carrying to the target, forexample, an agent coupled to the antibody. On the other hand, agentsthat suppress processes that tend to promote uncontrolled proliferationof cells (“antineoplastic agents”) can be delivered to target sites inthis manner.

The term “agent” is used herein in its broadest sense to refer to acomposition of matter, a process or procedure, a device or apparatusemployed to exert a particular effect. By way of non-limiting example, asurgical instrument may be employed by a practitioner as an “excising”agent to remove tissue from a subject; a chemical may be used as apharmaceutical agent to remove, damage or neutralize the function of atissue, etc. Such pharmaceutical agents are said to be “anticellular.”Cells may be removed by an agent that promotes apoptosis. A variety oftoxic agents, including other cells (e.g., cytotoxic T-cell lymphocytes)and their secretions, and a plethora of chemical species, can damagecells.

The term “by-stander”, as used herein, refers to a process or eventinitiated or affected by another, causative event or process.

The term “knockdown”, as used herein, refers to a method of selectivelypreventing the expression of a gene in an individual.

The term “oncogene”, as used herein, refers to any gene that regulates aprocess affecting the suppression of abnormal proliferative events.

The term “single nucleotide polymorphism” or “SNP”, as used herein,refers to a DNA sequence variation occurring when a single nucleotide inthe genome (or other shared sequence) differs between members of aspecies or between paired chromosomes in an individual. Singlenucleotide polymorphisms may fall within coding sequences of genes,non-coding regions of genes, or in the intergenic regions between genes.Single nucleotide polymorphisms within a coding sequence will notnecessarily change the amino acid sequence of the protein that isproduced, due to degeneracy of the genetic code. A Single nucleotidepolymorphism in which both forms lead to the same polypeptide sequenceis termed synonymous (sometimes called a silent mutation)—if a differentpolypeptide sequence is produced they are non-synonymous. Singlenucleotide polymorphisms that are not in protein-coding regions maystill have consequences for gene splicing, transcription factor binding,or the sequence of non-coding RNA.

The term “tissue array” or “tissue microarray”, as used herein, refersto high throughput platforms for the rapid analysis of protein, RNA, orDNA molecules. These arrays can be used to validate the clinicalrelevance of potential biological targets in the development ofdiagnostics, therapeutics and to study new disease markers and genes.Tissue arrays are suitable for genomics-based diagnostic and drug targetdiscovery.

As used herein, the term “shRNA” or “short hairpin RNA” refers to asequence of ribonucleotides comprising a single-stranded RNA polymerthat makes a tight hairpin turn on itself to provide a “double-stranded”or duplexed region. shRNA can be used to silence gene expression via RNAinterference. shRNA hairpin is cleaved into short interfering RNAs(siRNA) by the cellular machinery and then bound to the RNA-inducedsilencing complex (RISC). It is believed that the complex inhibits RNAas a consequence of the complexed siRNA hybridizing to and cleaving RNAsthat match the siRNA that is bound thereto.

As used herein, the term “RNA interference” or “RNAi” refers to thesilencing or decreasing of gene expression by siRNAs. It is the processof sequence-specific, post-transcriptional gene silencing in animals andplants, initiated by siRNA that is homologous in its duplex region tothe sequence of the silenced gene. The gene may be endogenous orexogenous to the organism, present integrated into a chromosome orpresent in a transfection vector that is not integrated into the genome.The expression of the gene is either completely or partially inhibited.RNAi inhibits the gene by compromising the function of a target RNA,completely or partially. Both plants and animals mediate RNAi by theRNA-induced silencing complex (RISC); a sequence-specific,multicomponent nuclease that destroys messenger RNAs homologous to thesilencing trigger. RISC is known to contain short RNAs (approximately 22nucleotides) derived from the double-stranded RNA trigger, although theprotein components of this activity are unknown. However, the22-nucleotide RNA sequences are homologous to the target gene that isbeing suppressed. Thus, the 22-nucleotide sequences appear to serve asguide sequences to instruct a multicomponent nuclease, RISC, to destroythe specific mRNAs. Carthew has reported (Curr. Opin. Cell Biol. 13(2):244-248 (2001) [16]) that eukaryotes silence gene expression in thepresence of dsRNA homologous to the silenced gene. Biochemical reactionsthat recapitulate this phenomenon generate RNA fragments of 21 to 23nucleotides from the double-stranded RNA. These stably associate with anRNA endonuclease, and probably serve as a discriminator to select mRNAs.Once selected, mRNAs are cleaved at sites 21 to 23 nucleotides apart.

As used herein, the term “siRNAs” refers to short interfering RNAs. Insome embodiments, siRNAs comprise a duplex, or double-stranded region,of about 18-25 nucleotides long; often siRNAs contain from about two tofour unpaired nucleotides at the 3′ end of each strand. At least onestrand of the duplex or double-stranded region of a siRNA issubstantially homologous to or substantially complementary to a targetRNA molecule. The strand complementary to a target RNA molecule is the“antisense strand”; the strand homologous to the target RNA molecule isthe “sense strand”, and is also complementary to the siRNA antisensestrand. siRNAs may also contain additional sequences; non-limitingexamples of such sequences include linking sequences, or loops, as wellas stem and other folded structures. siRNAs appear to function as keyintermediaries in triggering RNA interference in invertebrates and invertebrates, and in triggering sequence-specific RNA degradation duringposttranscriptional gene silencing in plants.

The term “xenograft”, as used herein, refers to the transfer ortransplant of a cell(s) or tissue from one species to an unlike species(or genus or family).

The term “orthotopic” or “orthotopic xenograft”, as used herein, refersto a cell or tissue transplant grafted into its normal place in thebody.

The term “fluorescent activated cell sorting” or “FACS”, as used herein,refers to a technique for counting, examining, and sorting microscopicparticles suspended in a stream of fluid. It allows simultaneousmultiparametric analysis of the physical and/or chemical characteristicsof single cells flowing through an optical and/or electronic detectionapparatus.

Generally, a beam of light (usually laser light) of a single wavelengthis directed onto a hydro dynamically focused stream of fluid. A numberof detectors are aimed at the point where the stream passes through thelight beam; one in line with the light beam (Forward Scatter, correlatesto cell volume) and several perpendicular to the beam, (Side Scatter,correlates to the inner complexity of the particle and/or surfaceroughness) and one or more fluorescent detectors. Each suspendedparticle passing through the beam scatters the light in some way, andfluorescent chemicals found in the particle or attached to the particlemay be excited into emitting light at a lower frequency than the lightsource. By analyzing the combinations of scattered and fluorescent lightpicked up by the detectors it is then possible to derive informationabout the physical and chemical structure of each individual particle.

The term “data mining”, as used herein, refers to the automated orconvenient extraction of patterns representing knowledge implicitlystored or captured in large databases, data warehouses, internetwebsites, other massive information repositories, or data streams.

The terms “over-express”, “over-expressing” and grammatical equivalents,as used herein, refer to the production of a gene product at levels thatexceed production in normal or control cells. The term “over-expression”or “highly expressed” may be specifically used in reference to levels ofmRNA to indicate a higher level of expression than that typicallyobserved in a given tissue in a control or non-transgenic animal. Levelsof mRNA are measured using any of a number of techniques known to thoseskilled in the art including, but not limited to Northern blot analysis.Appropriate controls are included on the Northern blot to control fordifferences in the amount of RNA loaded from each tissue analyzed, theamount of 28S rRNA (an abundant RNA transcript present at essentiallythe same amount in all tissues) present in each sample can be used as ameans of normalizing or standardizing the mRNA-specific signal observedon Northern blots. Over-expression may likewise result in elevatedlevels of proteins encoded by said mRNAs.

The term “heatmap”, as used herein, refers to a graphical representationof data where the values obtained from a variable two-dimensional mapare represented as colors. As related to the field of molecular biology,heat maps typically represent the level of expression of multiple genesacross a number of comparable samples as obtained from a microarray.

The term “phage display”, as used herein, refers to theintegration/ligation of numerous genetic sequences from a DNA library,consisting of all coding sequences of a cell, tissue or organism libraryinto the genome of a bacteriophage (i.e. phage) for high-throughputscreening protein-protein and/or protein-DNA interactions. Using amultiple cloning site, these fragments are inserted in all threepossible reading frames to ensure that the cDNA is translated. DNAfragments are then expressed on the surface of the phage particle aspart of it coat protein. The phage gene and insert DNA hybrid is thenamplified by transforming bacterial cells (such as TG1E. coli cells), toproduce progeny phages that display the relevant protein fragment aspart of their outer coat. By immobilizing relevant DNA or proteintarget(s) to the surface of a well, a phage that displays a protein thatbinds to one of those targets on its surface will remain while othersare removed by washing. Those that remain can be eluted, used to producemore phage (by bacterial infection with helper phage) and so produce anenriched phage mixture. Phage euted in the final step can be used toinfect a suitable bacterial host, from which the phagemids can becollected and the relevant DNA sequence excised and sequenced toidentify the relevant, interacting proteins or protein fragments.

The term “apoptosis”, as used herein, refers to a form of programmedcell death in multicellular organisms that involves a series ofbiochemical events that lead to a variety of morphological changes,including blebbing, changes to the cell membrane such as loss ofmembrane asymmetry and attachment, cell shrinkage, nuclearfragmentation, chromatin condensation, and chromosomal DNAfragmentation. Defective apoptotic processes have been implicated in anextensive variety of diseases; for example, defects in the apoptoticpathway have been implicated in diseases associated with uncontrolledcell proliferations, such as cancer.

The term “bioluminescence imaging” or “BLI”, as used herein, refers tothe noninvasive study of ongoing biological processes in livingorganisms (for example laboratory animals) using bioluminescence, theprocess of light emission in living organisms. Bioluminescence imagingutilizes native light emission from one of several organisms whichbioluminescence. The three main sources are the North American firefly,the sea pansy (and related marine organisms), and bacteria likePhotorhabdus luminescens and Vibrio fischeri. The DNA encoding theluminescent protein is incorporated into the laboratory animal eithervia a virus or by creating a transgenic animal. While the total amountof light emitted via bioluminescence is typically small and not detectedby the human eye, an ultra-sensitive CCD camera can imagebioluminescence from an external vantage point. Common applications ofBLI include in vivo studies of infection (with bioluminescentpathogens), cancer progression (using a bioluminescent cancer cellline), and reconstitution kinetics (using bioluminescent stem cells).

The term “consensus region” or “consensus sequence”, as used herein,refers to the conserved sequence motifs that show which nucleotideresidues are conserved and which nucleotide residues are variable whencomparing multiple DNA, RNA, or amino acid sequence alignments. Whencomparing the results of a multiple sequence alignment, where relatedsequences are compared to each other, and similar functional sequencemotifs are found. The consensus sequence shows which residues areconserved (are always the same), and which residues are variable. Aconsensus sequence may be a short sequence of nucleotides, which isfound several times in the genome and is thought to play the same rolein its different locations. For example, many transcription factorsrecognize particular consensus sequences in the promoters of the genesthey regulate. In the same way restriction enzymes usually havepalindromic consensus sequences, usually corresponding to the site wherethey cut the DNA. Splice sites (sequences immediately surrounding theexon-intron boundaries) can also be considered as consensus sequences.In one aspect, a consensus sequence defines a putative DNA recognitionsite, obtained for example, by aligning all known examples of a certainrecognition site and defined as the idealized sequence that representsthe predominant base at each position. Related sites should not differfrom the consensus sequence by more than a few substitutions.

The term “linkage”, or “genetic linkage,” as used herein, refers to thephenomenon that particular genetic loci of genes are inherited jointly.The “linkage strength” refers to the probability of two genetic locibeing inherited jointly. As the distance between genetic loci increases,the loci are more likely to be separated during inheritance, and thuslinkage strength is weaker.

The term “neighborhood score”, as used herein, refers to the relativevalue assigned to a genomic locus based on a geometry-weighted sum ofexpression scores of all the genes on a given chromosome, as ameasurement of the copy number status of the locus. A positiveneighborhood score is indicative of an increase in copy number, whereasa negative neighborhood score is indicative of a decrease in copynumber.

The term “expression score”, as used herein, refers to the expressiondifferences (i.e., the level of transcription (RNA) or translation(protein)) between comparison groups on a given chromosome. Theexpression score for a given gene is calculated by correlating the levelof expression of said gene with a phenotype in comparison. For example,an expression score may represent a comparison of the expressiondifferences of a given gene in normal vs. abnormal conditions, such asparental vs. drug-resistant cell lines. As used herein, the term“regional expression score” refers to the expression score of gene(s) inproximity to the locus in consideration. Since linkage strength betweengenetic loci decreases (i.e. decays) as the distance between themincreases, the “regional expression score” more accurately reflects theexpression differences between comparison groups by assigning greaterweight to the expression scores of genes in proximity to the locus inconsideration.

The terms “geometry-weighted” or “geometry-weighted sum”, as usedherein, refers to the significance attached to a given value, forexample an “expression score”, based on physical position, including butnot limited to genomic position. Since linkage strength between geneticloci decreases (i.e. decays) as the distance between them increases, the“weight” assigned to a given value is adjusted accordingly.

The term “copy number alteration” or “CNA”, as used herein, refers tothe increase (i.e. genomic gain) or decrease (i.e. genomic loss) in thenumber of copies of a gene at a specific locus of a chromosome ascompared to the “normal” or “standard” number of copies of said genethat locus. As used herein, an increase in the number of copies of agiven gene at a specific locus may also be referred to as an“amplification” or “genomic amplification” and should not be confusedwith the use of the term “amplification” as it relates, for example, toamplification of DNA or RNA in PCR and other experimental techniques.

The term “clonogenic assay”, as used herein, refers to a technique forstudying whether a given cancer therapy (for example drugs or radiation)can reduce the clonogenic survival and proliferation of tumor cells.While any type of cell may be used, human tumor cells are commonly usedfor oncological research. The term “clonogenic” refers to the fact thatthese cells are clones of one another.

The term “adjuvant therapy”, as used herein, refers to additionaltreatment given after the primary treatment to increase the chances of acure. In some instances, adjuvant therapy is administered after surgerywhere all detectable disease has been removed, but where there remains astatistical risk of relapse due to occult disease. If known disease isleft behind following surgery, then further treatment is not technically“adjuvant”. Adjuvant therapy may include chemotherapy, radiationtherapy, hormone therapy, or biological therapy. For example,radiotherapy or chemotherapy is commonly given as adjuvant treatmentafter surgery for a breast cancer. Oncologists use statistical evidenceto assess the risk of disease relapse before deciding on the specificadjuvant therapy. The aim of adjuvant treatment is to improvedisease-specific and overall survival. Because the treatment isessentially for a risk, rather than for provable disease, it is acceptedthat a proportion of patients who receive adjuvant therapy will alreadyhave been cured by their primary surgery. Adjuvant chemotherapy andradiotherapy are often given following surgery for many types of cancer,including colon cancer, lung cancer, pancreatic cancer, breast cancer,prostate cancer, and some gynecological cancers.

The term “matched samples”, as used herein, as for example “matchedcancer samples” refers to a sample in which individual members of thesample are matched with every other sample by reference to a particularvariable or quality other than the variable or quality immediately underinvestigation. Comparison of dissimilar groups based on specifiedcharacteristics is intended to reduce bias and the possible effects ofother variables. Matching may be on an individual (matched pairs) or agroup-wide basis.

The term “genomic segments”, as used herein, refers to any defined partor region of a chromosome, and may contain zero, one or more genes.

The term “co-administer”, as used herein, refers to the administrationof two or more agents, drugs, and/or compounds together (i.e. at thesame time).

The term “diagnose” or “diagnosis”, as used herein, refers to thedetermination, recognition, or identification of the nature, cause, ormanifestation of a condition based on signs, symptoms, and/or laboratoryfindings.

The term “resistance”, as used herein, refers to cancer cells that donot respond to chemotherapy drugs (i.e. chemotherapeutic agents).Typically, a first course of chemotherapy may prove highly beneficial,nearly annihilating a tumor, but a few resistant cancer cells oftensurvive and proliferate. Too often, despite more aggressive second andthird courses of chemotherapy, the remaining drug-defiant cells thrive,displaying increasing resistance to drug therapy and eventuallydisplaying virtual invulnerability to chemotherapy. After the drug'seffectiveness fades, the patient relapses. This occurs in patients witha variety of blood cancers and solid tumors, including breast, ovarian,lung, and lower gastrointestinal tract cancers. Nature Biotechnology18:IT18-IT20 (2000) [17]. Resistance to treatment with anticancer drugsresults from a variety of factors including individual variations inpatients and somatic cell genetic differences in tumors, even those fromthe same tissue of origin. Frequently resistance is intrinsic to thecancer, but as therapy becomes more and more effective, acquiredresistance has also become common.

The development of multidrug resistance (MDR) to chemotherapy remains amajor challenge in the treatment of cancer. Resistance exists againstevery effective anticancer drug and can develop by numerous mechanismsincluding decreased drug uptake, increased drug efflux, activation ofdetoxifying systems, activation of DNA repair mechanisms, andinsensitivity to drug-induced apoptosis. Methods Mol. Biol. 596:47-76(2010) [18].

In some embodiments, the present invention contemplates treating drugresistant cancer cells. It is not intended that the present invention belimited to the degree of resistance, i.e. resistance can be shown simplyby the fact that it takes higher doses of drug to kill these cells. Thecells need not be resistant at every dose. The cells may be resistantsuch that higher doses needed to kill the cells will not be welltolerated by the patient.

As used herein, “Doxorubicin” (trade name Doxil) also known as“hydroxydaunorubicin” or “Adriamycin” refers to a drug used in cancerchemotherapy, that is considered to be the most effective agent in thetreatment of breast cancer patients. Doxorubicin is an anthracyclineantibiotic, closely related to the natural product daunomycin, and likeall anthracyclines, work by intercalating DNA, with the most seriousadverse effect being life-threatening heart damage.

Doxorubicin is commonly used in the treatment of a wide range ofcancers, including some leukemia's and Hodgkin's lymphoma, as well ascancers of the bladder, breast, stomach, lung, ovaries, thyroid, softtissue sarcoma, multiple myeloma. It is frequently used in breast cancertherapy either as single-agent or in combination with other drugs likedocetaxel and cyclophosphamide. Unfortunately, resistance to this agentis common, representing a major obstacle to successful treatment. Mol.Cancer Ther. 5(8):2115-20 (2006) [19]. Doxorubicin is administeredintravenously, as the hydrochloride salt. It may be sold under the brandnames Adriamycin PPS, Adriamycin RDF, or Rubex. Commonly useddoxorubicin-containing regimens include, but are not necessarily limitedto, AC (Adriamycin, cyclophosphamide), TAC (taxotere, AC), ABVD(Adriamycin, bleomycin, vinblastine, dacarbazine), BEACOPP (bleomycin,etoposide, Adriamycin, cyclophosphamide, vincristine, procarbazine,prednisone), BEP (bleomycin, etoposide, platinum agent (cisplatin(Platinol)), CAP (cyclophosphamide, Adriamycin, fluorouracil (5-FU)),CAV (cyclophosphamide, Adriamycin, vincristine), CHOP (cyclophosphamide,Adriamycin, vincristine, prednisone), Ch1VPP/EVA (chlorambucil,vincristine, procarbazine, prednisone, etoposide, vinblastine,Adriamycin), CVAD/HyperCVAD (cyclophosphamide, vincristine, Adriamycin,dexamethasone), DT-PACE (dexamethasone, thalidomide, cisplatin orplatinol, Adriamycin, cyclophosphamide, etoposide), FAC (5-fluorouracil,Adriamycin, cyclophosphamide), m-BACOD (methotrexate, bleomycin,adriamycin, cyclophosphamide, Oncovin (vincristine), dexamethasone),MACOP-B (methotrexate, leucovorin (folinic acid), adriamycin,cyclophosphamide, Oncovin (vincristine), prednisone, bleomycin),ProMACE-MOPP (methotrexate, Adriamycin, cyclophosphamide,etoposide+MOPP), ProMACE-CytaBOM (prednisone, Adriamycin,cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine,methotrexate, leucovorin), VAD (vincristine, Adriamycin, dexamethasone),Regimen I (vincristine, Adriamycin, etoposide, cyclophosphamide) andVAPEC-B (vincristine, Adriamycin, prednisone, etoposide,cyclophosphamide, bleomycin).

Analogues of Doxorubicin for cancer chemotherapy include, but are notlimited to, daunorubicin, 4-demethoxydaunorubicin (idarubicin),pirarubicin (DaunoXome), epirubicin, pegylated liposomal doxorubicin(Lipo-Dox®), antibody-conjugated liposomal doxorubicin (e.g.S5A8-Lipo-Dox), 4′-epidoxorubicin, AD198,N-(5,5-Diacetoxypent-1-yl)doxorubicin, and Doxorubicin analogues 2-5,incorporating the following alkylating or latent alkylatingsubstituents, R, on the 3′-position of the daunosamine sugar. 2,R═NHCOC₆H₄(p)SO₂F; 3, R═NHCOCH2Br; 4, R═NHCOCH2Cl; 5,R═NHCON(NO)CH2CH2Cl. J Med Chem. 1991 February; 34(2):561-4 [20].

As used herein, “Ibrutinib”, also known as PCI-32765, refers to a drugfor the treatment of various types of hematopoietic related cancer, andis shown in FIG. 8B. However, in one embodiment, the present inventioncontemplates the use of ibrutinib for non-hematopoietic related cancers,and in particular for breast cancer.

As used herein, “AVL-292” also known as “Spebrutinib;” “CC-292;”“CC292;” “CC 292;” “AVL292;” “AVL-292;” and “AVL 292” isN-(3-((5-fluoro-2-((4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)phenyl)acrylamide, has CAS #: 1202757-89-8,and is shown in FIG. 8B.

DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated into and form a part ofthe specification, illustrate several embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The figures are only for the purpose ofillustrating a preferred embodiment of the invention and are not to beconstrued as limiting the invention.

FIG. 1A-C show growth inhibitory effects of BTK inhibitors on breastcancer cell lines.

FIG. 1A shows cell counts of MCF-10A, MCF7, SKBR3 and MDA-MB-231 cellstreated with vehicle, 1 μM and 10 μM of ibrutinib (Ibr) for 3 days.

FIG. 1B shows cell counts of SKBR3 and BT474 cells treated with vehicleand different concentration of ibrutinib, as indicated for 3 days.

FIG. 10 shows cell counts of BT474 cells treated with vehicle andibrutinb, AVL-292 and CGI-1746, as indicated concentration, for 3 days.Results are presented as percentage of control (vehicle). Error barsindicate the s.d. from three individual experiments, *p<0.05, **p<0.01compare with control.

FIG. 2A-D shows the effect of lapatinib and ibrutinib on cell growth andsignal transduction in BT474 cells.

FIG. 2A shows cell counts of BT474 cells treated with vehicle,lapatinib(Lap) and ibrutinib(Ibr) for 3 days. Results are presented aspercentage of control (vehicle). Error bars indicate the s.d. from threeindividual experiments, *p<0.05, **p<0.01 compare with control.

FIG. 2B shows BT474 cells were treated with laptanib and ibrutinib, asindicated, for 9 days on Matrigel culture condition.

FIG. 2C shows the alignment of EGFR family members with BTK.

FIG. 2D shows the effects of BTK inhibitors on EGFR family activation.BT474 cells were treated with different concentration of BTK inhibitorsibrutinib and AVL-292 or laptinib for 2 h. Whole cell lysates wereprepared for Westen blotting using antibody against p-EGFR, p-Her2,p-ERBB3, p-ERBB4, p-Akt, p-ERK protein. Anti-AKT and anti-ERK blottingwere done as loading controls.

FIG. 3A-D shows the effect of ibrutinib on cell cycle and apoptosis.

FIG. 3A shows the effect of ibrutinib and lapatinib on cell cycleprogression. BT474 cells were treated with the indicated concentrationof ibrutinib and lapatinib for 16 h. Cells were stained with propidiumiodide and analyzed by flow cytometry.

FIG. 3B show BT474 cells were treated with ibrutinib or lapatinib for 16h. Cell lysates were analyzed by western blotting using anti-cyclin DI,anti-p27 antibodies, anti-ERK as loading control.

FIG. 3C shows Bt474 cells were treated with ibrutinib or lapatinib for16 h. Apoptotic cells were identified by Alexa Fluor 488 Annexin V kit.*p<0.05, **p<0.01 compare with control.

FIG. 3D shows immunoblots showing apoptosis m BT474 cells afterindicated concentration of ibrutinib treatment for 16 h.

FIG. 4A-D shows inhibition pro-survival pathway re-activation byibrutinib.

FIG. 4A shows live cell count assay showing the NRG effects ondrug-treated BT474 cells (72 h), complete rescue, lapatinib (Lap) withNRG treatment; no rescue lapatinib, ibrutinib(Ibr) and ibrutinib withNRG treatment.

FIG. 4B shows crystal violet cell staining of BT474 or SKBR3 cellstreated with lapatinib (1 uM) with or without NRG1 (50 ng/ml), ibrutinib(1 uM) with or without NRG (50 ng/ml).

FIG. 4C shows immunoblots showing effects of NRG (50 ng/ml) on p-AKT anda-ERK after cells treated with lapatinib or ibrutinib (2 h) in BT474 andSkBR3 cells.

FIG. 4D shows a live cell count assay showing the NRG effects ondrug-treated BT474 cells (72 h), complete rescue, Lapatinib with NRG1treatment; no rescue, lapatinib alone or lapatinib and AVL with NRG1treatment.

FIGS. 5A&B shows BTK-C activation by Src in breast cancer cells.

FIG. 5A shows SKBR3-BTKC cells were treated with ibrutinib, LY294002 andSaracatinib as indicated concentration for 24 h. Cell lysates weretested for p-BTK, p-AKT and p-ERK. Anti-flag as loading control.

FIG. 5B shows SKBR3-btkc cells were treated with ibrutinib and differentconcentration of LY294002 for 24 h. Cell extration were tested forphosphorylation of the indicated protein. Anti-ERK as a loading control.

FIG. 6A-C show Ibrutinib inhibits Her2+ breast cancer cells growth invivo.

FIG. 6A shows images of tumors formed in animals after mammary fat padinjection of SKBR3 cancer cells. Animals were treated with vehicle,lapatinib (37.5 mg/kg or 75 mg/kg) or ibrutinib (6 mg/kg or 12 mg/kg)for 4 weeks.

FIG. 6B shows tumor growth curves obtained following fat pad injectionof SKBR3 cells. The data represent the mean±s.e. (n=5, *P<0.01).

FIG. 6C shows histological analysis of tumors from SKBR3 cells treatedwith ibrutinib(12 mg/kg) or vehicle. Shown are pHH3, caspase 3, pHer2,pBTK, pAkt and pErk staining.

FIGS. 7A&B shows positive correlation between BTK and Her2 expression insurgical specimens of human breast cancer tissue.

FIG. 7A shows Her2 positive and negative surgical specimens of humanbreast tumors (BR10010b) were subject to stain BTK and Her2. The datarepresent 50 of breast tumor specimens. Case 1 and case 2 representpositive staining BTK-C, easel and case 3 represent positive stainingHer2.

FIG. 7B shows a summary of the positive correlation. The correlationbetween the overexpression of BTK-C and Her2 was analyzed by Fisher'sexact test.

FIG. 8A shows the BTK-A & BTK-C kinase domain.

FIG. 8B shows the structures of PCI-32765 (Ibrutinib), CGI-1746,AVL-292, and lapatinib.

FIG. 8C shows that Ibrutinib and AVL-292 inhibit BTK-C activatingphosphorylation and are potentially useful as prostate cancer therapies.

FIG. 9A shows an experiment using EGF where cells are co-treated withEGF/lapatinib or EGF/ibrutinib.

FIG. 9B shows an experiment using EGF where cells are co-treated withEGF/lapatinib or EGF/ibrutinib.

DETAILED DESCRIPTON OF THE INVENTION

Tyrosine kinases (TKs) catalyze the reversible process of tyrosinephosphorylation, a key step in most signal transduction pathways thatgovern cellular proliferation, survival, differentiation, and motility.Dysregulation of TKs, as occurs through inappropriate expression,activation, or both, is commonly associated with human cancers(Blume-Jensen and Hunter 2001 [1]; Giamas, et al. 2010 [21]). As aresult, TKs, as a class, are the most commonly found dominant oncogenes(Baselga 2006 [22]; Blume-Jensen and Hunter 2001 [1]; Krause and VanEtten 2005 [23]; Vassilev and Uckun 2004 [24]).

Receptor protein tyrosine kinases (RPTKs) transmit extracellular signalsacross the plasma membrane to cytosolic proteins, stimulating theformation of complexes that regulate key cellular functions. Over halfof the 90 tyrosine kinases have been implicated in human cancers and arefor this reason considered highly promising drug targets. To gaininsight into the tyrosine kinases that contribute to breast cancerrelated cellular mechanisms, we carried out a large-scaleloss-of-function analysis of the tyrosine kinases, using RNAinterference, in the clinically relevant Erb-B2 positive, BT474 breastcancer cell line. The cytosolic, non-receptor tyrosine kinase Bruton'styrosine kinase (BTK), which has been extensively studied for its rolein B cell development, was among those tyrosine kinase genes requiredfor BT474 breast cancer cell survival. The BTK protein identified was analternative form containing an amino-terminal extension. Thisalternative form of the Btk message is also present in tumorigenicbreast cells at significantly higher levels than in normal breast cells.

Small molecules that directly inhibit the catalytic activity of tyrosinekinases have been sought as potential cancer chemotherapeutics. Recentsuccesses with a few well-studied tyrosine kinases have proven the valueof these proteins as drug targets. Imatinib mesylate (Gleevec) hasproven hugely successful in treating CML. The EGFR inhibitors, Gefitinib(Iressa) and erlotinib (Tarceva), are currently used on a variety ofsolid tumors (Krause and Van Etten 2005 [23]; Kris, et al. 2003 [25];Shepard, et al. 2008 [26]). Trastuzumab (Herceptin), a humanizedmonoclonal antibody that specifically inhibits Erb-B2, is widely used inthe treatment of breast cancers. Each of these treatments, however, hassignificant limitations related to tissue spectrum, acquired resistance,and efficacy in advanced disease (Nahta and Esteva 2006 [4]). Theidentification of additional TK genes and pathways that contribute tothe survival of distinct cancer cell types, so that they can beeffectively targeted, would be of great value.

Bruton's tyrosine kinase (BTK) is a key player in B cell development aswell as an important regulator of cell proliferation and cell survivalin various B cell malignancies. It has been reported that an isoform ofBTK (BTK-C) expressed in breast cancer protects these cells fromapoptosis. Herein, the effect of recently developed inhibitors of BTK onbreast cancer cells are tested. Inhibitors of BTK such as ibrutinib(PCI-32765), AVL-292 and CGI-1746 show reduction of the survival ofbreast cancer cell lines in vitro. It was found that ibrutinib treatmentsignificantly decreases the viability of HER2+ breast cancer cell linesin vitro at lower concentrations than the established breast cancertherapeutic lapatinib. It is thought that this may be due in part to theinhibition of EGFR family activation in addition to its effect on BTK-C.

Herein it is demonstrated that ibrutinib, but not AVL-292 and CGI-1746,efficiently blocks activation of EGFR, HER2, ErbB3, and ErbB4.Consequently, the activation of AKT and ERK signaling pathways are alsoblocked leading to an appreciable G1/S cell cycle delay, decreased cellproliferation and increased levels of apoptosis. NRG and EGF have beenshown to reactivate the AKT signaling pathway and promote the growthfactor-driven resistance that rescues HER2+ breast cancer cells from theantiproliferative effects of lapatinib. Ibrutinib, however, inhibits AKTre-activation by NRG or EGF. Consequently, HER2+ breast cancer cellproliferation remains blocked by ibrutinib even in the presence of thesefactors. Importantly, although AVL-292 has no effect on EGFR familyactivation, it prevents NRG- and EGF-dependent growth factor drivenresistance to lapatinib in HER2+ breast cancer cells suggesting that BTKactivity is important in this process. In vivo, ibrutinib inhibits SKBR3xenograft tumor growth. Immunofluorescence staining of tumor tissuesshows that ibrutinib blocks the phosphorylation of Her2, BTK, Akt andErk which result in decreased histone 3 phosphorylation and increasedcaspase-3 signals. Since it is also shown that BTK-C and HER2 are oftenco-expressed in human breast cancer, these observations indicate thatBTK-C is a potential therapeutic target and that ibrutinib could be aneffective drug, especially for HER2+ breast cancer.

INTRODUCTION

Bruton's tyrosine kinase (BTK) belongs to the TEC family, of cytoplasmictyrosine kinases [27]. It was identified in 1993 as a novel non-receptorprotein tyrosine kinase that is mutated in X-linked agammaglobulinaemia(XLA) [28, 29]. BTK is predominantly expressed in hematopoietic cellsincluding erythroid progenitors and myeloid cells [30]. BTK is acritical regulator of B cell receptor signaling. Studies haveestablished that BTK has a crucial role for B-cell development,differentiation, survival and signal transduction [31-33]. Due to itsrole in BCR signaling induced proliferation, BTK. has emerged as a noveltarget for the treatment of rheumatoid arthritis and other immunediseases. Recent studies have focused on the essential role of BTK. inmany B cell leukaemias and lymphomas [34, 35] which provides a rationalefor targeting the kinase in these malignancies. Second generation BTK.inhibitors including ibrutinib, AVL-292 and CGI-1746 were developed asimmunosuppressants and have been used in clinical trials for bloodmalignancies [36]. Recently, ibrutinib (Imbruvica) gained FDA approvalfor the treatment of mantle cell lymphoma, chronic lymphocytic leukemia,and Waldenstrom's macroglobulinemia [37]. An alternate isoform of BTK,BTK-C, was identified as a novel survival factor for breast cancer cellsin a large-scale loss-of-function analysis of human tyrosine kinasesusing an RNA interference library [38]. This study showed that althoughBTK is expressed at relatively low levels in several human breast cancercell lines and tumor tissues, it provides an essential functionprotecting breast cancer cells from apoptosis.

It has long been appreciated that HER2 is overexpressed or amplified intumors of about 20% of patients with early stage breast cancer andconfers an increased disease recurrence and a worse prognosis [39].HER2-directed therapies including trastuzumab, pertuzumab,ado-trastuzumab and lapatinib have been used in clinic and hassignificantly improved the outlook for patients with HER2-positivebreast cancer [40]. However, a significant proportion of these patientsstill relapses and succumbs to their disease [41]. Therefore, newclasses of drugs are needed, especially for HER2-positive advanced-stagebreast cancer and those that have developed resistance to currenttherapies.

Herein the effects of treating HER2-positive breast cancer cells with apotent, irreversibly-acting small molecule inhibitor of BTK, ibrutinibis described. It is shown that ibrutinib induces G1-S arrest andapoptosis in breast cancer cells. The effects of ibrutinib onHER2-positive breast cancer cells are shown to be not sensitive tostimulation with NRG1 or EGF as occurs with lapatinib. As the expressionof BTK-C and HER2 are positively correlated in surgical specimens ofhuman breast cancer tissues, these results indicate that ibrutinib is apotential therapy for this solid tumor type.

Results Activity of BTK Inhibitors in Human Breast Cancer Cells

In a functional genomic screen, a novel BTK isoform has been identifiedas a gene whose expression protects breast cancer cells from apoptosis[38]. In addition to genetic evidence, the first generation BTKinhibitor LFM-A13 was shown to increase apoptosis levels in breastcancer cells. The recently developed second generation BTK inhibitors,including ibrutinib, AVL-292 and CGI-1746, are more potent, morespecific and more useful clinically compared to LFM-A13 [42-44]. In2013, ibrutinib was approved by the FDA for treatment of B cellmalignancies [45, 46]. As a first step in exploring the potentialclinical utility of the second generation BTK inhibitors, cell growthassays were performed to determine the effect of these inhibitors onbreast cancer cells. It was found that ibrutinib results in decreasedcell number in breast cancer cells MCF7, SKBR3 and MDA-MB-231, but notin MCF-I0A cells (FIG. 1A). These results are consistent with previousfindings [38]. Surprisingly, it was observed that HER2-positive breastcancer cells SKBR3 are more sensitive to ibrutinib (1 μM), which reducescell numbers by more than 80% at 3 days (FIG. 1). To extend thesefindings to another HER2-positive breast cancer cell, the effect ofibrutinib on BT474 cell survival were tested. The result shows thatibrutinib inhibits HER2-positive breast cancer cell growth at aconcentration of 10 nanomolar (Figure IB). The IC50 for ibrutinib'seffect on HER2-positive breast cancer cells measured at 3 days ofculture is 0.03 μM. Also explored was whether HER2-positive breastcancer cells are sensitive to other BTK inhibitors. Although AVL-292 andCGI-1746 inhibit BTK-C kinase activity to the same degree as ibrutinib(FIG. 8A-C), they are less effective than ibrutinib causing a 30-40%decrease in cell numbers in HER2-positive breast cancer cell lines athigher concentrations (FIG. 1C). These results suggest that ibrutinibnot only inhibits BTK-C activity, but also affects other targetsspecifically required for HER2-positive breast cancer cell survival.

Ibrutinib Effects in HER2-Positive Breast Cancer Cells

Ibrutinib is a covalent inhibitor of BTK [47] that irreversibly binds toa cysteine residue (Cys-481) near the ATP binding pocket of BTK.Sequence alignments show that only 10 kinases in the human genome have acysteine residue at an analogous position. They include Blk, Btk, Bmx,EGFR, HER2, ErbB4, Itk, Jak3, Tee and Txk [48]. To compare theinhibitory effect of ibrutinib with lapatinib on HER2-positive breastcancer cells, their activity in monolayer culture was tested first.Lapatinib, a selective, reversible inhibitor of both EGFR and HER2 iscurrently used to treat HER2-positive breast cancer patients. Treatmentwith ibrutinib at different concentrations as indicated for 3 daysreduces BT474 cell number. 0.01 μM of ibrutinib reduces cell populationsto 30% of control (p<0.01). However, lapatinib, at the sameconcentration (0.01 μM) fails to reduce HER2-positive breast cancer cellnumbers significantly (FIG. 2A). A number of studies have shown thatcells' microenvironment can impact drug response [49, 50]. In order totest whether HER2-positive breast cancer cells are still sensitive toibrutinib in 3D matrigel culture condition, treatment of BT474 cellswith 0.1 μM or 0.5 μm of ibrutinib for 9 days significantly reduces cellnumber when compared with the control (FIG. 2B). Thus, it was found thatibrutinib reduces HER2-positive breast cancer cell number in bothmonolayer and 3D culture, and that ibrutinib has a more potent effect onHER2-positive breast cancer cells than lapatinib.

In HER2 overexpressing breast cancer cells, HER2 dimerizes with itspartner EGFR or Her3. HER2/Her3 heterodimers directly phosphorylate thep85 regulatory subunit of PI3K activating the PI3KJAkt pathway [51]. Inparallel, HER2/EGFR heterodimers also activate the MAPK pathway in mostcases [52]. To examine the effect of ibrutinib on levels of activatedAkt or Erk, BT474 cells was treated with 0.05 μM or 0.1 μM of ibrutiniband 0.1 μM of AVL-292 for 2 hours. It was found that ibrutinib inhibitsthe phosphorylation of EGFR, HER2, Her3 and ErbB4, which results inblocking downstream signaling that requires Akt or Erk activation.Compared with ibrutinib, AVL-292 does not block EGFR family signalingpathway activation, even though AVL-292 also covalently binds Cys481 onBTK. Lapatinib, a dual kinase inhibitor of EGFR and HER2, inhibits bothAKT and ERK phosphorylation (FIG. 2D). These results suggest thatibrutinib also serves as a pan-EGFR family inhibitor and blocks theactivation of each kinase, which leads to HER2-positive breast cancercells more sensitive to ibrutinib.

Ibrutinib Effects on Proliferation of HER2-Positive Breast Cancer Cells.

In HER2-positive breast cancer cells, the activation of HER2 stimulatesboth the MAPK and Akt signaling pathways, which results in cellproliferation due to increased G1-S phase transition and cell cycleprogression [53]. Treatment of Her-2 overexpressing BT474 human breastcancer cells for 24 hours with ibrutinib or lapatinib leads to anappreciable G1-S arrest. A significant 50% decrease in the number ofcells in the S phase of the cell cycle is observed at a concentration of0.03 μM for ibrutinib when compared with controls (FIG. 3A). Similarresults are seen for lapatinib [53]. This cell cycle delay is correlatedwith an increase in p27, an inhibitor of cell cycle progression, and adecrease in cyclinD1 (FIG. 3B).

An increase in the number of apoptotic cells following treatment with0.05 or 0.1 μM ibrutinib or lapatinib for 24 hours is observed. Theseresults are consistent with the results of earlier studies, which haveshown the effect of 0.1 or 0.5 μM lapatinib on cell survival [53].Compared with lapatinib, ibrutinib induces apoptosis 1.5 fold inHER2-positive breast cancer cells (FIG. 3C). The effect of ibrutinib oncell survival regulatory proteins was also examined. Ibrutinib blocksPLCγ1, PLCγ2, Akt and ERK phosphorylation, and increases cleaved PARP(FIG. 3D). Taken together, these results suggest that ibrutinibdecreases cell numbers by inducing both a G1-S delay and apoptosis inHER2-positive breast cancer cells.

Ibrutinib Blocks the Reactivation of AKT and ERK Pathways Induced byNRG1 or EGF in HER2-Positive Breast Cancer Cells.

Cancer cells typically express multiple receptor tyrosine kinases (RTKs)which control cell survival signals [54]. RTK ligands are produced viaautocrine tumor-cell production, paracrine tumor stroma production andsystemic production [55]. An increase in RTK ligands has been shown toresult in the resistance of cancer cells to RTK inhibitors [56]. InHER2-positive breast cancer cells, NRG1 is the most broadly activeligand, followed by EGF [57]. It was hypothesized that the greaterefficacy of ibrutinib may impact the occurrence of growth factordependent lapatinib resistance. The effect of exposing HER2-positivecells to 50 ng/ml of NRG1 with different concentration of lapatinib oribrutinib was first tested for 3 days. The results again show thatlapatinib or Ibrutinib potently suppresses cell growth. However, NRG1application is able to rescue lapatinib-induced growth inhibition inHER2-positive breast cancer cells allowing resistant cells to emergefrom the treatment (FIG. 4A). In contrast, HER2-positive breast cancercells cannot be rescued from ibrutinib-induced growth inhibition byNRG1. Similar results are observed using 50 ng/ml of EGF.

The ability of NRG1 or EGF signaling to rescue HER2-positive breastcancer cells from growth inhibition by HER2 kinase inhibitor lapatinibis consistent with other findings [56]. However, the findings thatneither NRG1 nor EGF was able to rescue HER2-positive breast cancercells from the growth inhibition by BTK inhibitor ibrutinib wereunexpected. To further confirm these findings, additional long-termco-treatment experiments were performed. Nine days lapatinib/NRGIco-treatment yields cells that exhibit lapatinib resistance in bothBT474 and SKBR3 cell populations. In contrast, 9-days ibrutinib/NRG1co-treatment does not yield ibrutinib resistant cells in HER2-positivebreast cancer cell lines (FIG. 4B). Similar results are observed inexperiments using EGF where cells are co-treated with EGF/lapatinib orEGF/ibrutinib (FIGS. 9A&B).

To investigate the signaling pathway which mediates the NRG1- orEGF-induced rescue of HER2-positive breast cancer cells, the activationof two downstream survival signaling pathways commonly engaged by RTKswas examined: the PI3K-AKT and MAPK pathway[57]. In the presence oflapatinib or ibrutinib alone, the activation of HER2, AKT and ERK areblocked. When cells are co-treated with NRG1/lapatinib, NRG1re-activates AKT without activation of HER2. Surprisingly, NRG1 cannotre-activate AKT under conditions of NRG 1/ibrutinib co-treatment (FIG.4C). These results suggest that the NRG1 rescuing effect is blocked byibrutinib which may target a second activated kinase. The results alsosuggest that lapatinib cannot block this second kinase.

The above results showed that ibrutinib blocks the re-activation of AKTin HER2-positive breast cancer cells induced by NRG1, whereas lapatinibdoes not. Since ibrutinib was originally designed to block BTKactivation and since BTK has been shown to activate AKT in B cells, thepossibility existed that BTK-C provided a pro-survival signal in thegenesis of lapatinib resistance. BTK-C was identified as a critical cellsurvival gene in a large scale RNAi screen in these cells was consistentwith this notion. For these reasons, it was hypothesized that BTK-Cmediated the re-activation of AKT induced by NRG1. However, sinceibrutinib is as potent as an inhibitor of EGFR family activation aslapatinib in HER2-positive breast cancer cells, there was a need todissociate the effects of EGFR inhibition from the effects of BTKinhibition. To test whether BTK-C is a secondary kinase that mediatesNRG1 rescue in HER2-positive breast cancer cells, the BT474 and SKBR3cells were treated with lapatinib, lapatinib/NRG1 or lapatinib/NRG1 plusAVL-292, a BTK inhibitor that does not inhibit the EGFR family (FIG.2A-D). It was found that NRG1 rescue is blocked by simultaneouslytargeting BTK and the EGFR family when cells treated with lapatinib andAVL-292 (FIG. 4D). These results provide evidence that BTK-C signalingis involved in the appearance of ligand-dependent lapatinib resistancein treated HER2-positive breast cancer cell populations.

BTK-C Signaling in HER2-Positive Breast Cancer Cells

The BTK signaling pathway has been extensively studied in hematopoieticcells. Upon antigen binding to the BCR, PI3K is activated. PI3K activityrecruits BTK to the cell membrane through a PIP3-PH domain interaction,which allows SYK and LYN to fully activate BTK [58-60]. In previousstudies, it was shown that a novel isoform of BTK (BTK-C) is expressedin human breast cancer cell lines and tissues. To explore the signalingactivation of BTK-C in breast cancer cells, two potential upstreamregulatory molecules of BTK-C: PI3K and Src were assessed [61]. First,the SKBR3-BTK-C cells were treated for 24 hours with establishedconcentrations of PI3K inhibitor LY294002 (5 or 10 μM) or Src inhibitorSarcatinib (5 or 10 μM). The phosphorylation of BTK-C is completelyblocked by Sarcatinib at 10 μM [62]. The phosphorylation of AKT, as adownstream target of BTK-C, also decreases. In contrast, 10 μM ofLY294002 does not suppress BTK-C activation (FIG. 5A). Since thepossibility exists that this lower concentration of LY294002 may notblock BTK-C activation, the concentration of LY294002 was increased to50 μM and repeated the test. The results show that LY294002 at 50 μMcompletely blocks AKT activation, but not BTK-C activation (FIG. 5B).Collectively, these results suggest that Src, or a closely relatedkinase, is the main upstream molecule of the BTK-C activation signalingpathway in HER2-positive breast cancer cells. It also suggests that, dueto the presence of an additional domain adjacent to the pleckstrinhomology domain, the BTK-C isoform is not activated through PIP3interaction as occurs with the B cell version of the kinase.

Effects of Ibrutinib Treatment on HER2-Positive Breast Tumor XenograftsGrowth in Vivo

These molecular experiments carried out in vitro pointed to thepossibility that ibrutinib treatment might be useful in inhibiting Her2positive tumor progression. To test this possibility, the effect ofibrutinib on xenografts of SKBR3 in NOD/SCID mice was assessed.Ibrutinib treatment inhibits tumor growth when administered to animalsbetween 6 mg/kg/day and 12 mg/kg/day. At day 28, tumor volumes in micethat received 12 mg/kg are 45% smaller than the volumes in mice thatreceive a vehicle control (p<0.01)(FIG. 6A). To determine whetherinhibition of tumor growth was correlated with inhibition of the targetmolecules, Her2 and BTK phosphorylation were examined in tumor tissuesby immunofluorescence staining as well as downstream targets AKT and ERKphosphorylation. Phosphorylation of Her2, BTK, AKT and ERK are inhibitedby ibrutinib treatment (FIG. 6B). In addition, changes in proliferationand apoptosis markers in tumor tissues reflect the effect that ibrutinibhas in xenografts of SKBR3. The ability of ibrutinib to target both Her2and BTK-C in these xenografts confirms the in vitro findings andprovides a strong rationale for the use of ibrutinib in HER2-positivebreast cancer chemotherapy.

The Expression of BTK-C in HER2-Positive Breast Cancer Tissue

The above results suggest that simultaneously targeting HER2 and BTKrepresents an improved approach to the treatment of HER2-positive breastcancer. The BTK-C isoform was identified as a gene whose function iscritical for breast cancer cell survival even though its expressionlevels are quite low. For this gene product to be a useful target, it isnecessary to determine the frequency of its expression in HER2+ breastcancer. Although currently routine for many genes, establishing BTK-Cexpression patterns in tumors by querying available databases is notpossible. The BTK-C isoform has only recently been described and littleis understood regarding its expression. This is due in part to the factthat Affymetrix probes for this region have only been included in exonmicroarrays very recently. Additionally, the BTK-C isoform encodes theentire B-cell version sequence (BTK-A) and is annotated as a 5′ UTRsplice variant of BTK-A [38]. To provide further evidence about theexpression of BTK-C on breast cancer tissues especially on HER2-positivebreast cancer tissues, the co-expression of BTK-C and HER2 in surgicalspecimens of human breast cancer tumor tissues by immunofluorescence wasexamined. Again, it was found that BTK-C expresses in 30% of breastcancer tumor tissues (FIGS. 7A&B). Among 23 HER2-positive human breastcancer tissues, 43% are positive for BTK-C. There is a statisticallysignificant association of expression of BTK-C with HER2 expression(p<0.01) (FIG. 7B). These findings strongly suggest that BTK-C and HER2are positive correlation expression in human breast cancer tissues, andibrutinib could be a new drug for treatment HER2 breast cancer bytargeting BTK and HER2 simultaneously.

DISCUSSION

Previous findings showed that a novel isoform of BTK (BTK-C) isfrequently expressed in human breast cancer cells and tissues and thatthis isoform plays a crucial role in cell survival. Consistent withthese findings, it was reported that several second generation BTKinhibitors reduce breast cancer cell number in a variety of cell typesincluding MCF7, MDA-MB-231 and SKBR3 cells. Importantly, it was foundthat HER2-positive breast cancer cells are highly sensitive toibrutinib. Ibrutinib, with an IC50 for these cells of 0.03 μM, is muchbetter at decreasing cell numbers than the other BTK inhibitors, AVL-292and CGI-1746, and compares favorably with lapatinib. In vitro, enzymaticactivity assays show that ICS0 of lapatinib for EGFR and HER2 are 10.8and 9.2 nM respectively. These results indicate that the sensitivity ofHER2-positive breast cancer cells to ibrutinib is due to the drug'sability to simultaneously suppress the activation of both BTK-C as wellas the EGFR family. Consistent with previous studies, ibrutinibtreatment results in induction of a pronounced G1/S delay and increasedapoptosis in HER2-positive breast cancer cells [53]. It was also foundthat BTK-C plays an essential role in the ability of HER2-positivebreast cancer cells to develop resistance to lapatinib under conditionswhen the growth factors NRG1 or EGF are present. NRG1 or EGF are unableto re-activate PI3K/Akt or MAPK signaling pathways and allow cells toescape the inhibitory effects of lapatinib when BTK-C is blocked byAVL-292, which is incapable of inhibiting the EGFR family. Sinceibrutinib inhibits the activity of the EGFR family and inhibits theBTK-dependent reactivation of the PI3K/Akt or MAPK signaling pathways,it is a strong candidate to replace lapatinib as a combination therapywith trastuzumab for HER2-positive breast cancer patients who mightotherwise develop resistance to lapatinib. As was found that theexpression of BTK-C and HER2 are positively correlated in human breastcancer tissue with 40% of HER2-positive breast cancer tissues also beingBTK-C positive, the potential utility of the drug is significant.

In accord with the in vitro studies, these results show that ibrutinibtreatment at 12 mg/kg/day causes a significant 45% inhibition ofHer2-positive tumor growth in vivo. Animals tolerate ibrutinib at thisdose, although ibrutinib-related toxicity was observed. Consistent withibrutinib's antitumor effects, staining of the proliferation marker,phosphorylated histone H3, decreased in treated xenografts tumor tissuescompared with untreated control tumor tissues. Moreover, focal apoptoticlesions caused by ibrutinib treatment was observed, as evidenced bycaspase 3 staining in xenografts tumor tissues. In these experiments,lapatinib treatment at 75 mg/kg/day was less potent in SKBR3 tumorxenograft models than ibrutinib treatment at 12 mg/kg/day. This islikely due to the ability of ibrutinib to target irreversibly both BTKand EGFR family members and block re-activation of PI3K/AKT or MAPKcaused by growth factors such as NRG1 in vivo. Taken together, thesefindings show that ibrutinib actively blocks BTK and EGFR familyactivation efficiently inhibiting HER2-positive breast cancer growth invivo. As a result, this study supports the use of ibrutinib as aHER2-positive breast cancer treatment and indicates that targeting BTK-Cwith second generation BTK inhibitors such as ibrutinib or AVL-292 mayavert the development of drug resistance in breast cancer patients.

The advent of HER2-directed therapy has significantly improved theprognosis of patients with metastatic and early HER2-positive breastcancer. Currently, there are two classes of HER2-targeting drugs used inthe clinic. Trastuzumab, Trastuzumab-DM1 and Pertuzumab are antibodybased drugs, whereas lapatinib is a small molecule that reversiblyinhibits both EGFR and HER2, blocking the PBK/Akt and MAPK pathways.Lapatinib is the only small compound used to treat HER2-positive breastcancer that has metastasized to the brain due to its ability to crossthe blood-brain barrier. Despite this recent progress, acquiredresistance to HER2-directed therapy still results in relapse andprogression of HER2-positive disease. The primary mechanism ofresistance to lapatinib stems from increased levels of EGFR or HER3ligands such as NRGI or EGF in the tumor microenvironment whichre-activate the PI3K/Akt and MAPK signaling pathways that are blocked bythe drug [56]. The activation of the signaling pathway downstream ofEGFR family members other than HER2 is dependent on heterodimerizationof the EGFR family member triggered by NRG1 or EGF binding to theextracellular ligand-binding domain. Increased expression of HER2 at thecell membrane leads to constitutive signaling of downstream pathways:PI31(/Akt and the Ras/Raf/MEI( )MAPK, which are involved in regulatingcell growth, survival, differentiation, migration and metastasis.Reactivation of these pathways allows cells to escape from theanti-proliferative and anti-apoptotic effects of the HER2 inhibitors.

Ibrutinib was the first reported covalent inhibitor of BTK [48]. As anorally bioavailable, selective and irreversible inhibitor, ibrutinib hasbeen undergoing multiple clinical trials targeting treatment of variousB-cell malignancies and has shown promising clinical efficacy [47].Based on sequence alignments, 10 kinases in the human genome have anorthologous cysteine residue [48]. The EGFR family includes EGFR/Her1,HER2, Her3 and Her4 all of which are involved in several aspects oftumorigenesis. These kinases include EGFR, HER2 and Her4 covalently bindto the thiol group of Cys481 in the ATP pocket region of BTK (positionCys5I 5 of BTK-C). In vitro, enzymatic activity assays show that andhave IC50 of ibrutinib for each enzyme in the nm range BTK, EGFR, HER2and Her4 are 0.5, 5.6, 9.4 and 0.6 nM respectively. In addition,ibrutinib also inhibits ITK, an essential enzyme in Th2 T cells, whichshifts the balance between Th1 and Th2 T cells and potentially enhancesantitumor immune responses [63]. Given the relevance of this spectrum oftargets, it is therefore not surprising that ibrutinib is effective indecreasing the proliferation and increasing apoptosis in HER2-positivebreast cancer cells.

Based on BTK expression data, its function in breast cancer cells andibrutinib's targets, it can predicted that ibrutinib, an irreversibleBTK inhibitor, might be used in neoadjuvant or adjuvant setting withTrastuzumab or other chemotherapeutic compounds for HER2-positive breastcancer in the future. Moreover, ibrutinib abolishes the ability ofeither NRG1 or EGF to re-activate Akt or Erk in HER2-positive breastcancer cells. In vivo, ibrutinib blocks HER2-positive breast cancer cellxenograft growth. Finally, a strong correlation of the co-expression ofBTK-C and HER2 in human breast cancer tissues was identified. This workdemonstrates that BTK-C could be a novel therapeutic target for breastcancer, and that current second-generation BTK inhibitors could be usednew drugs in HER targeted therapy for HER2-positive breast cancerpatients.

Materials and Methods Cell Culture and Chemicals.

Breast cancer cell lines and MCF-10A were obtained from American TypeCulture Collection. All cell lines were cultures in DMEM (Hyclone)supplemented with 10% fetal bovine serum (FBS; Hyclone) and 100 units/μlof penicillin-streptomycin (Cellgro), except for MCF-10A cells culturedas indicated in. The BTK inhibitors Ibrutinib was purchased fromChemieTek, AVL-292 was purchased from MedKoo Biosciences, CGI-1764 waspurchased from Axon Medchem. Recombinant human-β1 and Matrigel werepurchased from R&D. Src inhibitor Saracatinib was purchased fromSelleckchem. LY294002 was purchased from Cell Signaling.

Cell Viability Assays.

For live cell counts, cell growth on 96-well plates were fixed 4%formaldehyde and counterstained with Hoechst 33342 for nuclei. Images ofcells were acquired using an In Cell Analyzer 1000 (GE Healthcare)high-content imaging system. At least 30 fields were imaged per well.Statistics were performed using the In Cell Investigator 3.4 imageanalysis software (GE Healthcare).

3D cell culture was performed as previously described [64]. The BT474cells were propagated in DMEM with FBS. Single cells in mediumcontaining 5% Matrigel were seeded at a density of 5×10⁴ cells/cm² on aMatrigel-coated well. The top medium with 5% Matrigel and ibrutinib orlapatinib was changed every 3 days. Using 1 μM of ethidium bromidestains cell death during 3d culture.

Cells were cultured in 6-well plates for indicated days. Cells werefixed with 3.7% paraformaldehyde for 10 min and washed with PBS. Afterwash, cells were stained with 0.05% CV for 30 min. Whole plate waswashed with tape water. Dry plate was added with 0.5 ml methanol tosolubilize the dye. Taking an aliquot to 96-well plate and reading CVstain with OD 540. Tumorospheres were counted under low magnification.

Cell Cycle Analysis

Cells were cultured in six-well plates and treated with vehicle,lapatinib or ibrutinib at the concentration as indicated for 16 hours.After trypsinized, cells were collected and washed with cold PBS. Cellswere fixed with 70% ethanol, washed and stained with PI/Triton X-100staining solution (0.1% triton X-100, 2μg/ml PI and 0.2 mg/ml DNAse-freeRNAse) for 30 min. Samples were analyzed by flow cytometry.

Immunofluorescence:

Human breast cancer tissue sections (BR10010b, US Biomax, Inc. MD) werebaked for 1 h at 62° C., serial alcohol for rehydration and microwavedin 0.0 IM sodium citrate for 20 min for antigen retrieval. The sectionswere serum blocked for 30 min, incubated overnight at 4° C. with firstantibodies in phosphate-buffered saline and subsequently withCy5-labeled secondary antibodies for 60 min, nucleus were stained withHoechst 33342. The stained sections were mounted with anti-fade solutionfor microscopy. The two by two tables for human data was analyzed byFisher's Exact Test. Significance were determined by the alpha level of0.5.

Immunoblotting

Immunoblotting was performed essentially as described previously. Equalamounts of proteins were used. Antibodies used were anti-EGFR (1:1000),anti-HER2 (1:1000), anti-ERBB3, ERBB4 antibody, anti-BTK, anti-AKT, ERK,anti-PLCγ1, PLCγ2 and anti-PARP (1:1000, cell signaling), anti-Flagantibody (1:1000, Sigma), anti-rabbit IgG-HRP and mouse IgG-HRP (1:5000,Jackson ImmunoResearch) [65].

Apoptosis Assay

Apoptotic cells were assessed with Alexa Fluor 488 annexin V apoptosisKit (Invitrogen). Cells were treated with lapatinib of ibrutinib for 24hours. Cells were trypsinized and washed with cold PBS and resuspend thecells in 1× annexin-binding buffer to 1×10⁶ cells/ml. Add 5p1 AlexaFluro 488 annexin V and 1 μl 100 μg/ml PI to each 100p1 of cellsuspension. Incubate the cells for 15 min at room temperature. After theincubation, add 400 μ1 1× annexin-binding buffer, mix gently and keepthe sample on ice. Samples were analyzed on a BD LSR II Flow Cytometer(BD Biosciences, San Jose, Calif.). The data were analyzed using theFlowJo software package (Treestar Inc., Ashland, Oreg.).

Animal Experiments

NOD/SCID mice were purchased from Jackson Lab (The Jackson Laboratory,Bar Harbor, Me., USA). All mouse procedures were approved by the AnimalCare and Use Committees of SUNY Albany and performed in accordance withinstitutional policies.

For xenograft tumor studies, 1×10⁶ SKBR3 cells were suspended in 50 μlMatrigel (BD Biosciences) diluted 1:2 with DMEM and injected intomammary fat pad. Treatment began when tumors were palpable. Ibrutinibwas given p.o. 6 mg/kg/day or 12 mg·kg·day in a vehicle of 1% DMSO/30%polyethylene glycol/1% Tween 80. Lapatinib was given p.o. 75 mg/kg/dayor 37.5 mg/kg/day. The tumor volume in mm³ is calculated by the formula:volume=(width)2×length/2 every 7 days.

Thus, specific compositions and methods of Bruton's tyrosine inhibitorshave been disclosed. It should be apparent, however, to those skilled inthe art that many more modifications besides those already described arepossible without departing from the inventive concepts herein. Moreover,in interpreting the disclosure, all terms should be interpreted in thebroadest possible manner consistent with the context. In particular, theterms “comprises” and “comprising” should be interpreted as referring toelements, components, or steps in a non-exclusive manner, indicatingthat the referenced elements, components, or steps may be present, orutilized, or combined with other elements, components, or steps that arenot expressly referenced.

Although the invention has been described with reference to thesepreferred embodiments, other embodiments can achieve the same results.Variations and modifications of the present invention will be obvious tothose skilled in the art and it is intended to cover in the appendedclaims all such modifications and equivalents. The entire disclosures ofall applications, patents, and publications cited above, and of thecorresponding application are hereby incorporated by references.

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We claim:
 1. A method of treating cancer, comprising: a) providing i) asubject with cancer, ii) a inhibitor of a tyrosine kinase, and iii) anEGFR inhibitor, and b) treating said subject with said inhibitors. 2.The method of claim 1, wherein said cancer is breast cancer.
 3. Themethod of claim 2, wherein said breast cancer is HER2+ breast cancer. 4.The method of claim 1, wherein said tyrosine kinase is Bruton's TyrosineKinase.
 5. The method of claim 1, wherein said tyrosine kinase is avariant of Bruton's Tyrosine Kinase comprising an amino-terminalextension.
 6. The method of claim 1, wherein said EGFR inhibitor islapatinib.
 7. The method of claim 1, wherein said EGFR inhibitor isselected from the group consisting of gefitinib, erlotinib, cetuximab,panitumumab, and vandetanib.
 8. The method of claim 1, wherein saidtyrosine kinase inhibitor is selected from the group consisting ofibrutinib (PCI-32765), AVL-292 and CGI-1746.
 9. The method of claim 1,wherein said treating said subject with said inhibitors is sequential.10. The method of claim 1, wherein said treating said subject with saidinhibitors is simultaneous.
 11. The method of claim 1, wherein treatingwith said inhibitors results in reduced proliferation of at least someof said cancer cells within said subject.
 12. A method of treatingcancer, comprising: a) providing i) a subject with cancer and ii)AVL-292, iii) lapatinib, and b) treating said subject with said AVL-292and lapatinib.
 13. The method of claim 12, wherein said cancer is breastcancer.
 14. The method of claim 13, wherein said breast cancer comprisesHer-2 positive cells.
 15. The method of claim 12, wherein said treatingsaid subject with said inhibitors is sequential.
 16. The method of claim12, wherein said treating said subject with said inhibitors issimultaneous. 17-18. (canceled)
 19. A pharmaceutical anticancercomposition comprising AVL-292 and lapatinib.
 20. (canceled)